M Gerin

H Liszt

CO + as a probe of the origin of CO in diffuse interstellar clouds

Keywords:

CO + as a probe of the origin of CO in diffuse interstellar clouds

M. Gerin, H.

Introduction

The diffuse interstellar medium (ISM) hosts a rich chemistry where many species reach molecular abundances similar to those in denser and darker regions despite the lower densities (a few tens to a few hundred particles per cm -3 ) and the relatively unattenuated far-ultraviolet (FUV) illumination by the interstellar radiation field (Snow & McCall 2006;Gerin et al. 2016;Liszt et al. 2014aLiszt et al. ,b, 2018;;Gerin et al. 2019). Diffuse and translucent interstellar clouds (Snow & McCall 2006) have long been used as testbeds of interstellar chemistry (Glassgold & Langer 1975, 1976;Black & Dalgarno 1977;Van Dishoeck & Black 1986;van Dishoeck & Black 1988). These efforts were successful in explaining the measured column densities for H 2 and small trace molecules such as CH, OH, and CN using chemical reactions at temperatures 30 -80K corresponding to kinetic temperatures measured in H 2 (Savage et al. 1977), but in some cases assuming regions of density > ∼ 1000 cm -3 along the line of sight. However, the observed column densities of CO and the common presence of high column densities of CH + presented challenges to such models of thermal, cosmic-ray, and UV-driven ion-chemistry in diffuse molecular gas.

The observed CO column densities are empirically explained by the serendipitous detection of unexpectedly high column densities of HCO + seen in absorption at 89.2 GHz (Lucas & Liszt 1996). A nearly constant abundance of HCO + relative to H 2 , X(HCO + ) = N(HCO + )/N(H 2 ) = 3 × 10 -9 ± 0.2dex is found in comparison with OH and CH (Gerin et al. 2019), using their fixed abundances relative to H 2 established through direct absorption observations at optical and UV wavelengths (Sheffer et al. 2008;Weselak et al. 2009Weselak et al. , 2010)). With such significant column densities, the observed HCO + ions will recombine with ambient thermal electrons at densities n(H) > ∼ 100 cm -3 to produce the observed CO column densities (Liszt & Lucas 2000;Visser et al. 2009).

The gas phase chemistry developed during the 1970s-1980s era was qualitatively correct in predicting a fixed abundance ratio N(HCO + )/N(OH) via a dominant chemical chain that proceeds from C + + OH → CO + + H (Glassgold & Langer 1975, 1976;Dagdigian 2019) to CO + + H 2 → HCO + + H and HCO + + e - → CO + H. However, the observed N(HCO + )/N(OH) ≈ 1/30 is roughly thirty times higher than the predicted value through this simple chemical scheme. Some HCO + can also be produced in this chemistry by the reaction of C + and H 2 O, of which the abundance is 28% of that of OH (Gerin et al. 2016;Wiesemeyer et al. 2016). Both reactions, CO + + H 2 and C + + H 2 O, produce equal amounts of HCO + and the slightly less tightly bonded isomer HOC + that is observed with N(HOC + )/N(HCO + ) = 0.015±0.003 (Gerin et al. 2019). This disparity in the abundances of HCO + and HOC + is much too large to be explained by the isomerization reaction H 2 + HOC + → H 2 + HCO + as discussed below.

To reproduce the observed HCO + abundance at the low mean densities of diffuse clouds, the most successful models include transient production mechanisms that involve dynamical processes in magnetized gas such as low-velocity shocks or turbulent vortices, in which ions and neutral species are partially decoupled (Godard et al. 2014;Lesaffre et al. 2020). The resulting slow velocity drift can therefore be used as an additional energy source to drive endothermic chemical reactions that could not operate otherwise (Sheffer et al. 2008;Visser et al. 2009), espe-cially the reaction C + + H 2 → CH + + H, which is endothermic by 0.36 eV or about 4300 K. These models therefore include a specific chemical pathway to CO, which starts from CH + and is followed by a series of rapid hydrogen abstraction reactions producing CH + 2 and CH + 3 . These two ions react with atomic oxygen to produce the required HCO + , and not HOC + . In this scheme, HCO + is once again the precursor of CO, but the predicted CO and HCO + abundances fit the observations well, and the relative rarity of HOC + arises very naturally.

Therefore, the relative abundances of the three molecular ions CO + , HCO + , and HOC + provide key information concerning the mechanisms at the origin of CO in diffuse gas. Despite the pivotal role played by CO + in some versions of the chemistry, its abundance relative to H 2 is much less well constrained than those of HCO + and HOC + . CO + was first identified in the interstellar medium toward the bright photodissociation region (PDR) M17SW and the young, high-excitation planetary nebula NGC 7027 (Latter et al. 1993). CO + is now routinely observed in dense PDRs such as the Orion Bar (Goicoechea et al. 2017) or MonR2 (Treviño-Morales et al. 2016). In such regions, CO + is located at the very edge close to the HI/H 2 transition, and its abundance relative to HCO + ranges from one to several tens of percent (Fuente et al. 2003). In dense PDRs, the intense FUV radiation can pump H 2 into vibrationally excited levels that can reach a significant population (Agúndez et al. 2010). The internal energy of this vibrationally excited H 2 can trigger the endothermic reaction of C + + H 2 forming CH + , as shown by the widespread distribution of CH + emission in such regions (Goicoechea et al. 2019). Paradoxically, the presence of CO + in dense PDRs is a sign that nonthermal chemical processes are working, while nonthermal processes are invoked in models of diffuse molecular gas to remove the need to produce CO + on the path to CO.

There are no reported detections of CO + in the diffuse and translucent interstellar medium: in this paper, we present observations with the Atacama Large Millimeter Array (ALMA) that constrain the abundance of CO + there for the first time. The observation strategy is presented in Section 2, and the results are discussed in Section 3 together with the implications for the CO chemistry that are emphasized in Sections 4 and 5. The conclusions are summarized in Section 6.

Spectroscopy and observations

Spectroscopy

Absorption spectroscopy represents the best method to probe the molecular content of diffuse clouds, because the molecular excitation is weak at the low densities (a few tens to a few hundred cm -3 ) and pressure (p/k ≈ 3 × 10 3 -10 4 K-cm -3 ) of these regions (Jenkins & Tripp 2011a;Gerin et al. 2015;Goldsmith et al. 2018), and the level populations are concentrated in the lowest rotational levels. As a 2 Σ molecular ion, the energy levels of CO + are described by three quantum numbers; N, the rigid body angular momentum quantum number; S = 1/2, the electron spin angular momentum quantum number; and J = N +S = N ±1/2, the total angular momentum quantum number (Sastry et al. 1981).

The CO + ground state rotational transitions at 117.7 GHz and 118.1 GHz are close to a strong atmospheric line from molecular oxygen at 118.75 GHz, rendering their observation from the ground very difficult. Hence, observations of CO + have generally targeted excited transitions where the sky transmission is much better. We chose to search for the N = 2 → 1 transitions because the frequencies of this first excited transi- Notes.

( a) The spectroscopic data were extracted from the Cologne Database for Molecular Spectroscopy (CDMS; Müller et al. (2001Müller et al. ( , 2005)); Endres et al. (2016)). (b) For an excitation temperature of 2.73 K. (c) For an excitation temperature of 5 K. tion near 236 GHz are easily accessible from ground-based radio observatories, and the level population remains significant even in diffuse interstellar gas. This N = 2 → 1 transition is split into three hyperfine components, with the strongest component at 236.06 GHz. Table 1 lists the three targeted transitions, their frequencies, Einstein A-coefficient, and the conversion factor between the integrated line opacity and the molecule column density N(CO + )/ τ dv assuming that the energy-level population is determined by the cosmic microwave background at 2.73 K.

Apart from CO + , the setting of the ALMA correlator included spectral windows targeting the 13 CO(2 -1), C 18 O(2 -1) and H 2 CO(3 0,3 -2 0,2 ) transitions that were accessible with the same receiver tuning while including a dedicated continuum spectral window for phase calibration. Properties of the CO lines are given in Table 1, where the column density/optical depth conversion is calculated for an excitation temperature of 5 K that is typical for this more easily excited species in diffuse molecular gas (Goldsmith et al. 2018).

Target directions

The targeted sources are J1717-3342 and J1744-3116, two bright quasars situated at small galactic latitude behind the Galactic bulge that were known to have high intervening column densities of Galactic neutral gas detected in molecular absorption (Gerin & Liszt 2017;Liszt & Gerin 2018;Riquelme et al. 2018). Their positions, continuum flux densities S ν , channel-channel baseline rms line/continuum noise and line profile integrals, and velocity intervals used here as integration intervals for the targeted CO + transitions are given in Table 2. Comparable quantities for the 13 CO(2-1) and C 18 O(2-1) lines are given in Table 4.

ALMA observations

The observations were performed with ALMA Band-6 receivers tuned near 236 GHz during ALMA Cycle 7 under the project code 2019.1.00120.S. The CO + lines were observed with a channel spacing of 244 kHz, corresponding to a velocity resolution of 0.31 kms -1 . The channel spectral resolution is twice the channel spacing. As in our earlier observations toward these sources, the bandpass calibrator was fixed to J1924-2914. Absorption spectra were extracted from the standard pipeline-processed data prod- The CO + spectra were shifted vertically and multiplied by 10. HCO + is shown in black, CO + in red, and 13 CO(2-1) in blue for comparison. The HCO + data are taken from Liszt & Gerin (2018) and Gerin & Liszt (2017). ucts at the peak of the continuum map in each spectral window.

The ALMA spectra of 13 CO, CO + , and HCO + are displayed in Fig. 1. The H 2 CO (3 0,3 -2 0,2 ) line was detected in both directions and will be discussed in a future paper.

Other data discussed here

Also shown in Figures 1 and 2 are 89.2 GHz ALMA HCO + J=1-0 absorption line profiles with 0.205 km s -1 channel spacing and 0.41 km s -1 spectral resolution from our earlier works (Gerin & Liszt 2017;Liszt & Gerin 2018). Profile integrals are quoted in Table 3, assuming excitation in equilibrium with the CMB and using N(HCO + ) = 1.12×10 12 cm -2 τ dv as before. Also shown in Fig. 2 are the nearest λ2.6mm CO J=1-0 emission profiles with an 8 .8 beamsize from the work of Bitran et al. (1997) and a λ21cm H I emission profile from Kalberla et al. (2005) taken with a FWHM beam of 0.6 • . The CO emission profile for J1744-3116 at l,b = 357.875 • ,-1 • is very nearly along the same sightline; for J1717-3342, the CO profile at 352.75 • ,2.5 • is centered 6 .6 away.

Results and discussion

3.1. The sight lines toward J1717-3342 and J1744-3116

Absorption spectra of HCO + , 13 CO, and CO + toward both sources are shown in Fig. 1, and Fig. 2 presents a comparison of the absorption spectra of CO and HCO + with emission spectra of HI and 12 CO(1-0) described in Section 2.4. Column densities of HCO + , 13 CO, C 18 O, and H 2 (N(H 2 ) = N(HCO + )/3 × 10 -9 ) are given in Tables 3 and4. To compute N( 13 CO) and N(C 18 O), we assumed an excitation temperature of 5 K (Table 1), which is consistent with the observed brightness of the 12 CO emission that is seen toward or near these sources, 1 -2.5 K.

The diffuse nature of the gas along these lines of sight has previously been discussed by Gerin & Liszt (2017), Liszt & Gerin (2018), and Riquelme et al. (2018). High gas column densities accumulate over long paths through the Galactic disk at low galactic latitude, even without encountering dense clouds. This is indicated in our new data by the large ratio of 13 CO and C 18 O integrated optical depths or column densities, N( 13 CO)/N(C 18 O) = 29±5 toward J1744-3116 (Table 4). If the oxygen and carbon ratios have the usual interstellar values, 16 O/ 18 O ∼ 520 and 12 C/ 13 C ∼ 65 (Wilson & Rood 1994;Milam et al. 2005;Keene et al. 1998;Giannetti et al. 2014), a fully molecular gas would have N( 13 CO)/N(C 18 O) = 8 toward J1744-3116. The much larger value we observe arises through a combination of selective photodissociation of C 18 O, and, more b Upper limits are 3σ.

likely, fractionation of 13 CO through endothermic carbon isotope exchange when the majority of the free gas phase carbon is in C + (Liszt 2017). Our value for N( 13 CO) toward J1744-3116 in Table 4 agrees with that given by Riquelme et al. ( 2018) based on J=1-0 absorption, N( 13 CO) = 6.3 × 10 15 cm -2 .

In our earlier work we also derived carbon isotope ratios H 12 CO + /H 13 CO + = 58 ± 9 and 64 ± 4 in the low-velocity gas toward J1717-3342 and J1744-3116, respectively. These values are typical of Galactic disk gas near the Sun and quite different from what is observed inside the Galactic bulge. Therefore, although the sight lines employed here cross the Galactic bulge, the low-velocity gas that is analyzed in CO and CO + resides relatively nearby in the disk and is discussed under the assumption that local conditions are applicable.

Overall, the CO(1-0) emission is spread over the same velocity range as the HCO + absorption. Toward J1744-3116, there is a fair correspondence of the main CO emission peaks with the HCO + absorption features, but the HCO + absorption profiles are more complex. The positive velocity component toward J1744-3116 is detected in 13 CO(2-1) and C 18 O(2-1) absorption and is associated with a self-absorption feature in the H I spectrum that was discussed in Liszt & Gerin (2018). The presence of the three CO isotopologues indicates that this velocity component probably has the largest extinction and mean density and could be associated with translucent rather than diffuse gas. There is no self-absorption in the HI profile coincident with the negative velocity component, but the main 13 CO(2-1) absorption is associated with a faint C 18 O(2-1) feature.

The CO(1-0) emission differs more from the HCO + absorption toward J1717-3342, where the direction of the emission spectrum is 6.6 away. Of the eight main HCO + absorption features, only one is detected in 13 CO(2-1), and none in C 18 O(2-1), showing that the gas is more diffuse along this line of sight. The CO emission is weaker than toward J1744-3116, barely reaching 1 K. As discussed by Liszt et al. (2010), the CO(1-0) emission is highly variable in diffuse gas as it traces the regions where CO reaches column densities above N(CO) = 10 15 cm -2 that are high enough to produce detectable emission. This transition occurs over a small range of H 2 column densities and depends on the local physical conditions, but CO emission at the level of 1 K-km s -1 integrated intensity is not observed in regions where the molecular fraction of H-nuclei in H 2 is much below 0.6.

The comparison of the emission and absorption profiles therefore demonstrates that the two observed lines of sight encounter matter with variable physical conditions, ranging from diffuse to translucent gas. The upper limits for the CO + integrated opacities shown in Table 2 provide constraints on the CO + column densities and abundances for the whole range of physical conditions. For the diffuse molecular gas sampled in absorption the molecule excitation is dominated by the cosmic microwave background because of the low gas densities. Therefore, the CO + column densities have been derived assuming an excitation tem-perature of 2.73 K. They are reported in Table 3 together with the HCO + column densities in the same velocity intervals. The achieved upper limit on CO + column densities leads to a very low value of about 0.04 for the N(CO + )/N(HCO + ) ratio. The corresponding upper limit on the CO + abundance relative to H 2 is X(CO + ) < 1.2 × 10 -10 , using the standard value for the HCO + abundance relative to H 2 in the diffuse molecular gas, 3 × 10 -9 (Lucas & Liszt 1996;Gerin et al. 2019). These values are of a factor a few times higher than those established for HOC + , N(HOC + )/N(HCO + ) = 0.015, and X(HOC + ) = 0.45 × 10 -11 (Gerin et al. 2019).

CO + as a source of HCO +

Despite the excellent sensitivity with achieved fractional rms flux density levels ∆S ν /S ν of 0.22% and 0.83% for J1717-3342 and J1744-3116, respectively, CO + was not detected toward these sources. The derived 3σ upper limits N(CO + )/N(HCO + ) < ∼ 0.04 and N(CO + )/N(H 2 ) < 1.2 -1.3 × 10 -10 are similar for both sources in Table 2, because J1717-3342, with a smaller intervening column density, is much brighter in the continuum. The achieved upper limit on the relative abundance is somewhat above the values seen in dense PDRs where emission lines from CO + are detected and X(CO + ) reaches a few times 10 -11 . They are below the relative abundances seen in the exceptional case of the young planetary nebula NGC 7027, where N(CO + )/N(H 2 ) = 5 × 10 -9 (Fuente et al. 2003).

It is straightforward to show that the relative abundance of CO + is too small to provide a major source of the observed HCO + in diffuse molecular gas, even independent of the HCO + abundance. In chemical terms, the reaction CO + + H 2 → HCO + + H forming HCO + can be compared with the rate at which HCO + recombines with electrons with rates as given in Table 5.

Equating formation and recombination of HCO + , neglecting photodissociation that is four orders of magnitude slower according to the Kinetic Database for Astrochemistry (KIDA) (Wakelam et al. 2015):

k 2 n(CO + )n(H 2 ) = α(T ) n(HCO + )n(e),
and when rearranging, we have n(CO + )/n(HCO + ) = (α(T )/k 2 )n(e)/n(H 2 ), or equivalently

n(CO + )/n(HCO + ) = (2/f H 2 ) (α(T )/k 2 ) x(e),
where f H 2 is the fraction of H-nuclei in H 2 , x(e) is the electron fraction, n(H 2 ) is the density of H 2 molecules, and n(H) is the total density of H-nuclei. Taking x(e) = 2 × 10 -4 corresponding to the observed free gas-phase carbon abundance

Source

N(HCO + ) N(CO + ) N(CO + )/N(HCO + ) N(CO + )/N(H 2 ) a 10 12 cm -2 10 12 cm -2 J1717-3342 7.49 ± 0.03 < 0.30 < 0.040 < 1.2 × 10 -10 J1744-3416 23.35 ± 0.06 < 0.98 <0.042 < 1.3 × 10 -10 a N(H 2 ) = N(HCO + )/3 × 10 -9 . n(C + )/n(H) = 1.6 × 10 -4 (Sofia et al. 2004) with a small added contribution from cosmic ray ionization of atomic hydrogen in largely molecular gas, the required amount of CO + needed to restore the HCO + lost to recombination is n(CO + )/n(HCO + ) = (0.6/f H 2 ) × (40/T ) 0.69 . This is some 25 times higher than our upper limit if f H 2 = 0.6 and T = 40 K. We note above that CO emission, even at the low levels shown in Fig. 2, does not arise in regions of very small molecular fraction and at temperatures high enough to trigger the formation of CH + : these are only characteristic of regions of strong turbulent energy dissipation.

Alternatively, equating the HCO + formation and recombination rates once again but setting n(HCO + )/n(H 2 ) = 3 × 10 -9 and x(e) = 2 × 10 -4 = 2 × 10 -4 (2n(H 2 )/f H 2 ), for the required relative abundance of CO + one derives

X(CO + ) = (1.8 × 10 -9 /f H 2 ) × (40/T ) 0.69 .
This is comparable to the CO + relative abundance seen in NGC 7027 (∼ 5 × 10 -9 , Fuente et al. (2003)) and is at least 15 times above the upper limit achieved here, 1.2 × 10 -10 , if T = 40 K (Table 2). In this simplified analysis, reducing X(CO + ) below our upper limit would require temperatures of about 2000 K, which correspond to the order of magnitude achieved in the turbulence-driven chemistry, which can only be present in small and intermittent regions of space and time, where other thermal and nonthermal processes are operating. CO + is responsible for producing at most a few percent of the HCO + that recombines with ambient thermal electrons to form CO in diffuse molecular gas.

The thermal chemistry of CO + , HOC + , and HCO +

Our upper limits show that CO + cannot be responsible for forming the observed CO in diffuse molecular gas, because CO + does not exist in a sufficient quantity to replenish HCO + as it recombines with ambient electrons. However, beyond that, our limits combined with the observed relative abundance N(HOC + )/N(HCO + ) = 1.4 ± 0.2 × 10 -4 (Gerin et al. 2019) can be used to gain further insights into the thermal chemistry that is working in diffuse molecular gas. Most of the reactions that form HCO + also form HOC + , yet their observed abundances differ by a factor of 70. To explore this, we extended the thermal CO + and HOC + chemistry discussed by Gerin et al. (2019) to include some hypotheticals corresponding to uncertainties in reaction rates in the KIDA reaction rate database (Wakelam et al. 2015) and molecular abundances (Gerin et al. 2016). As discussed in Gerin et al. (2019), some of the rates of the reactions controlling the formation of CO + and HCO + were not well known, especially the reaction C + + OH → CO + + H/CO + H + and the isomerization reaction destroying HOC + and forming HCO + , HOC + + H 2 → HCO + + H 2 . The rate of this reaction is reported as 4 × 10 -10 cm 3 s -1 at 25 K and 300 K (Smith et al. 2002), which is about 1/5 the Langevin capture value, and, in this temperature range, it is probably independent of the temperature.

As originally suggested by Glassgold & Langer (1975, 1976), CO + can be formed by the reaction of C + with OH, and also by the reaction of O with CH + . CO + is destroyed by recombination with electrons and in chemical reactions with atomic and molecular hydrogen, and photo-dissociated with a free space rate 1 × 10 -10 s -1 that is always at least 300 times slower than the other destruction rates in the calculations discussed here. The OH relative abundance to H 2 , X(OH) = 1.0 ± 0.14 × 10 -7 varies only narrowly and is well-determined in local diffuse clouds (Weselak et al. 2009(Weselak et al. , 2010) ) such as those probed in this work. The excellent correspondence of the OH and H 2 O absorption line profiles and of the H 2 O and HCO + profiles along long sight lines across the Galactic plane (Wiesemeyer et al. 2016;Gerin et al. 2019) further indicates that this value of the OH abundance holds for the Galactic disk as well. The rate of the reaction of OH and C + was recently calculated by Dagdigian (2019), providing accurate information on the rate coefficient and branching ratio over a temperature range 10 -1000 K. We approximated the rate constant for C + + OH forming CO + as k = 2.2 × 10 -9 (300/T) 0.107 cm 3 s -1 (see Table 5 ). By contrast, the abundance of CH + is locally variable, and CH + may not co-exist with the other species if it is predominantly formed in the nonthermal chemistry (Godard et al. 2014;Valdivia et al. 2017). We take X(CH + ) as a free parameter with X(CH + ) = 10 -9 by default in the following discussion.

HOC + can be formed in the reaction of H 2 with CO + and in the reaction of C + with H 2 O, of which we take the fixed relative abundance X(H 2 O) = 2.4 × 10 -8 from Gerin et al. (2016). We ignored HOC + formation by the reaction of O with CH 3 + because the formation of CH 3

+ is an aspect of the nonthermal chemistry. HOC + recombines with electrons and is susceptible to photodissociation, but most importantly is isomerized to HCO + in reaction with H 2 . The rate constant for the isomerization reaction of H 2 and HOC + has alternatives in the KIDA database as discussed previously. Here, we considered the two extreme values for the rate constant, 10 -11 cm 3 s -1 given for 10-280 K and 4.7 × 10 -10 cm 3 s -1 given at 305 K.

The CO + and HOC + chemistry is simple when other molecular abundances are held fixed, but it involves the thermal and ionization balance for the temperature-sensitive recombination rates and the atomic/molecular hydrogen balance for various reactions with CO + and HOC + . To address this, we performed calculations similarly to those described earlier to account for the observations of HF and CF + (Liszt et al. 2015). In brief, we calculated the self-consistent global atomic/molecular, ionization and thermal equilibrium in gas spheres of constant total hydrogen number and column density, subject to the usual interstellar radiation fields. The hydrogen column density was varied, and molecular abundances were integrated along the central line of sight to form the results that are displayed here. Plotting the results against N(H 2 ) removes some model sensitivities; for instance, to the impact parameter about the cloud center used for the line of sight integration. Fig. 3. Models of thermal chemistry for the relative abundances N(CO + )/N(HCO + ) and N(HOC + )/N(HCO + ) with default rates to the left, and deviations in the middle and right panels. In all panels, results for HOC + are shown in green and for CO + in orange. In all cases, N(HCO + ) is taken to be the observed value N(HCO + ) = 3 × 10 -9 N(H 2 ). Left panel: Baseline models with the observed upper limit for N(CO + )/N(HCO + ) from this work and the mean N(HOC + )/N(HCO + ) ratio from Gerin et al. (2019) are illustrated schematically as horizontal dashed lines. Middle panel: The solid lines represent the case that the rate constant of H 2 + HOC + → HOC + +H 2 interconversion is taken as 10 -11 cm 3 s -1 . The dashed-dotted lines represent model results when the rate constant for the reaction H 2 + CO + → HOC + + H is set to 0. Right panel: The solid lines show models in which the reaction of C + and H 2 O does not form HOC + and the dashed-dotted lines show model results when CH + is as abundant as OH, X(CH + ) = 10 -7 .

The results are shown in Fig. 3. To the left is a baseline model using default values (Table 5). The model produces CO + relative abundances about 2.5 times smaller than our observed upper limits, and about twice as much HOC + as observed. Declines in the abundances of CO + and HOC + for very high N(H 2 ) arise from the recombination of the free gas-phase carbon from C + to neutral carbon and CO. In the middle panel, the solid lines show results when H 2 -catalyzed HOC + → HCO + isomerization has the rate constant 10 -11 cm 3 s -1 , a factor of 2.5 above the default value. The green curve for HOC + shows that the predicted abundance of HOC + grows even further above what is observed when the HOC + → HCO + interconversion is slow. The orange curves show what happens when the rate constant for HOC + formation via H 2 + CO + → HOC + + H is set to 0. The predicted amount of CO + grows well above our upper limits, and the abundance of HOC + falls far below the observed mean. We conclude that HOC + is forming mostly via C + + OH followed by CO + + H 2 , but much or most of the HOC + produced by these chemical reactions is lost to isomerization into HCO + .

In the right panel of Fig. 3, the solid curves show model results when the reaction of C + and H 2 O does not form HOC + : all of the observed HOC + can indeed be made by the reaction of C + and OH, and agreement with the observed HOC + /HCO + ratio is somewhat better when the contribution from H 2 O is neglected. However, H 2 O is observed to be present with X(H 2 O) = 2.4 × 10 -8 (Gerin et al. 2016) and would be expected to form some amount of HOC + . The default model may predict an overabundance of CO + or perhaps some aspect of the reaction of C + and H 2 O is not fully understood: a slower rate or a branching ratio that favors HCO + would lead to a lower contribution of this reaction to the HOC + formation.

Moreover, CH + may not co-exist with the other species if it is predominantly formed in the nonthermal chemistry (Godard et al. 2014;Valdivia et al. 2017) and X(CH + ) is negligible (10 -9 ) in the default model. The final deviation from the default model is represented by the dash-dot lines in the right-hand panel showing results when X(CH + ) = X(OH) = 10 -7 is large. The reaction of O + CH + becomes an important source of CO + in the thermal chemistry when X(CH + ) > ∼ 5 × 10 -8 and our upper limits (Green et al. 2019) derived by equating CO formation and photo-destruction for sight lines with HCO + and CO column densities measured in absorption (Liszt & Lucas 1998;Liszt et al. 2019), as discussed in Section 5. on CO + by themselves imply X(CH + )/X(OH) < ∼ 3. At such high CH + abundances the discrepancy in HOC + grows even larger.

Conditions for the growth of CO

Balancing the formation of CO through the dissociative recombination of HCO + and the destruction of CO by photodissociation can constrain the physical conditions where CO might form in diffuse molecular gas. With a free-space photodissociation rate k d = 2.43 × 10 -10 s -1 for the Draine radiation field (defined as G 0 = 1) and an attenuation scaling as e -γA V , this balance locally implies

n(CO) n(HCO + ) = α(T )x(e)n(H) G 0 k d e -γA V .
For a uniform slab, integrating the attenuation term from 0 up to half the slab extinction,

+ hν → C + + O 1.04 × 10 -10 KIDA N(CO) N(HCO + ) = α(T )x(e)n(H) G 0 k d 0.5γA V 1 -e -0.5γA V .
n(H)/G 0 can therefore be expressed as

n(H)/G 0 = N(CO) N(HCO + ) k d (1 -e -0.5γA V ) α(T )x(e)0.5γA V ,
where we approximate A V = 3.1E(B-V) and take E(B-V) from Green et al. (2019), replacing local quantities with lineof-sight averages. The error bar in E(B-V) is a nominal value 0.02 mag. We take γ = 3.88 and use the reaction rates and rate coefficients given in Table 5. Figure 4 presents the variation of n(H)/G 0 as a function of the reddening E(B-V) for the existing sample of quasars sight lines with HCO + and CO column densities determined in absorption (Liszt & Lucas 1998;Liszt et al. 2019). We used conditions as described in Section 2, a uniform temperature of 40K, and a uniform electron fraction x(e) = 2 × 10 -4 .

The mean value for the sample is < n(H)/G 0 >∼ 300 cm -3 . For a standard interstellar radiation field of G 0 = 1, the calculated densities n(H) ∼ 100 -300 cm -3 are modest for E(B-V) > ∼ 0.5 mag, even in the absence of any consideration of CO shielding by itself or by H 2 . The higher densities n(H) ∼ 1000 -2000 cm -3 calculated at E(B-V) < ∼ 0.2 mag are higher by a factor of ∼ 5 than those inferred from the brightness of HCO + emission in these directions (Liszt 2020). Thermal pressures p/k ∼ 40 K × 1000 cm -3 are an order of magnitude higher than typical values in the diffuse molecular gas (Jenkins & Tripp 2011b) and would more likely represent the conditions under which a turbulencedriven chemistry forms HCO + . A lower value of the radiation field toward these sight lines would bring the derived densities closer to the mean values. The data shown in this plot include the sight lines with CO and HCO + detections, which may bias the derived n(H/G 0 to somewhat high values as a higher density implies a faster CO production rate.

The inverse variation of n(H)/G 0 with E(B-V) in Fig. 4, mirroring the functional dependence on Av, reflects the fact that the observed values of N(CO)/N(HCO + ) are not correlated with E(B-V) and show a moderate scatter of at most a factor of 1. A majority of the selected sight lines have N(CO)/N(HCO + ) between 1000 and 2000. We also plotted n(H)/G 0 against the molecular fraction f H 2 = 2N(H 2 )/N(H) taking N(H 2 ) = N(HCO + )/3 × 10 -9 and N(H) = a× E(B-V). Requiring f H 2 ≤ 1 in all cases implies a > ∼ 8 × 10 21 Hnuclei cm -2 mag , but there is no trend for n(H)/G0 to vary with f H 2 calculated in this way.

Summary and conclusions

We sought to test the origin of CO in diffuse molecular gas within the generally accepted framework that CO is predominantly formed through the dissociative electron recombination of HCO + . HCO + is widely observed in diffuse molecular gas, with a relative abundance N(HCO + )/N(H 2 ) = 3 × 10 -9 .

In a quiescent, thermal, cosmic-ray, and UV-driven chemistry, HCO + is predominantly formed in the reactions C + + OH → CO + + H and CO + + H 2 → HCO + + H. Using Cycle 7 ALMA Band-6 observations at 236 GHz (Table 1), we searched for CO + absorption toward two bright compact extragalactic millimeterwave continuum sources seen at low latitude in the inner Galaxy and known to be occulted by high column densities of HCO +bearing diffuse molecular gas (see Section 2 and Table 2). We failed to detect CO + at levels sufficient to demonstrate in Section 3 that the reaction CO + + H 2 → HCO + + H cannot replenish more than a few percent of the observed HCO + that recombines. The non-detection of CO + confirms that HCO + is mainly produced in the reaction between oxygen and carbon hydrides CH + 2 or CH + 3 induced by suprathermal processes, while CO + and HOC + result from reactions between C + and OH and H 2 O in the thermal chemistry that occurs in quiescent diffuse molecular gas. The observed CO + and HOC + abundances relative to H 2 set an upper limit on the rate coefficient of these reactions.

In Section 4, we have explored the coupled thermal chemistries of CO + and HOC + given our observational upper limits on CO + and existing observations of HOC + showing N(HOC + )/N(HCO + ) = 0.015 ± 0.003. A baseline cosmic-ray and UV-driven thermal chemical model overproduced HOC + by a factor of two, but by as much as a factor of six if the isomerization reaction HOC + + H 2 → HCO + + H 2 does proceed rapidly. The option of a low rate for this reaction in the KIDA database should probably be ignored. The predicted HOC + abundance is in better agreement with the observations if the reaction of C + and H 2 O preferentially forms HCO + , rather than HOC + . We placed a weak limit on the relative abundance of CH + in quiescent gas, N(CH + )/N(OH) < ∼ 3 or X(CH + )/X(H 2 ) < ∼ 3 × 10 -7 , such that the reaction O + CH + does not become a significant source of CO + , substantially overproducing it.

In Section 5, we have derived characteristic number densities n(H) at which CO would form in diffuse molecular gas with observed abundances. For E(B-V) > ∼ 0.5 mag, the densities are modest, n(H) ∼ 100 -300 cm -3 , but for CO-bright regions of low extinction E(B-V) ∼ 0.1 -0.2 mag they are an order of magnitude higher n(H) ∼ 1 -2 × 10 3 cm -3 , and such heavily over-pressured gas is more properly considered in the context of a turbulent dissipation chemistry. The presence of CO in sight lines of low reddening could also indicate a lower radiation field intensity.

Fig. 1 .

 1 Fig.1. Absorption spectra observed toward J1717-3342 (left) and J1744-3116 (right). The spectra were normalized by the continuum flux density. The CO + spectra were shifted vertically and multiplied by 10. HCO + is shown in black, CO + in red, and 13 CO(2-1) in blue for comparison. The HCO + data are taken fromLiszt & Gerin (2018) andGerin & Liszt (2017).

Fig. 2 .

 2 Fig.2. Comparison of the HCO + (1-0) (black) and 13 CO (2-1) (green) absorption with the 12 CO (blue) and scaled HI (gray) emission. J1717-3342 is shown at the top and J1744-3116 at the bottom. C 18 O(2-1) is also shown in orange for J1744-3116.

Fig. 4 .

 4 Fig. 4. Number density n(H)/G vs. reddening E(B-V)(Green et al. 2019) derived by equating CO formation and photo-destruction for sight lines with HCO + and CO column densities measured in absorption(Liszt & Lucas 1998;Liszt et al. 2019), as discussed in Section 5.

Table 1 .

 1 Spectroscopic parameters of observed lines.

	Transition a	Frequency	A	N(CO + )/ τ dv b
	N, J	MHz	s -1	cm -2 km -1 s
	2,3/2 -1 3/2 235380.046 0.689 × 10 -4	1.01 × 10 14
	2,3/2 -1 1/2 235789.641 3.465 × 10 -4	2.00 × 10 13
	2,5/2 -1 3/2 236062.553 4.172 × 10 -4	1.12 × 10 13
	Transition a	Frequency	A	N( 13 CO)/ τ dv c
	J	MHz	s -1	cm -2 km -1 s
	2 -1	220398.684 6.076 × 10 -7	2.42 × 10 15
	Transition a	Frequency	A	N(C 18 O)/ τ dv c
	J	MHz	s -1	cm -2 km -1 s
	2 -1	219560.354 6.012 × 10 -7	2.42 × 10 15

Table 2 .

 2 Summary of CO + observations. Velocity interval used to integrate the line profile.

	Source	l	b	LSR Velocity a	Line	Flux density S ν	σ l/c	τ dv b
		o	o	km s -1		Jy		km s -1
	J1717-3342 352.7333 2.3911 J1744-3116 357.8635 -0.9968	-14..10 -20..20	CO + J=5/2-3/2 CO + J=3/2-1/2 CO + J=5/2-3/2 CO + J=3/2-1/2	0.780 0.780 0.234 0.251	0.0027 <0.027 0.0034 <0.033 0.0084 <0.087 0.0107 <0.111
		a

Table 3 .

 3 HCO + and CO + column densities.

Table 4 .

 4 CO isotopologue J=2-1 observations and column densities.

	Source	Line	Flux	σ l/c	τ dv	N a
			Jy		km s -1	10 14 cm -2
	J1717-3342 13 CO 0.818 0.0024 0.260(0.004)	6.3(0.4)
		C 18 O 0.822 0.0123	< 0.055	< 1.3
	J1744-3116 13 CO 0.244 0.0073 2.975(0.023) 72.0(0.6)
		C 18 O 0.244 0.0084 0.102(0.018)	2.5(0.4)
			a Upper limit is 3σ.

Table 5 .

 5 Rates and rate coefficients.

	Reaction	Symbol	Rate Coefficient	Reference
			cm -3 s -1
	C + + OH → CO + + H CO + + H 2 → HCO + + H C + + H 2 O → HCO + + H C + + H 2 O → HOC + + H HOC + + H 2 → HCO + + H 2 HOC + + H 2 → HCO + + H 2 HCO + + e -→ CO + H	k 1 k 2 k 3 k 4 k 5 k 6 α(T )	2.2 × 10 -9 (300/T) 0.107 2.1 × 10 -9 2.1 × 10 -9 2.1 × 10 -9 4 × 10 -10 1 × 10 -11 1.43 × 10 -5 /T 0.69	Dagdigian (2019) KIDA KIDA KIDA, Martinez et al. (2008) Smith et al. (2002) KIDA Hamberg et al. (2014)
	Reaction	Symbol	Rate	Reference
			s -1
	CO + hν → C + O CO +	k d	2.43 × 10 -10 exp(-3.88A V )	KIDA, Heays et al. (2017)

Acknowledgements. The authors thanks the referee Helmut Wiesemeyer for his thoughtful comments and suggestions. This paper makes use of the following ALMA data: ADS/JAO.ALMA#2019.1.00120.S.. ALMA is a partnership of ESO (representing its member states), NSF (USA) and NINS (Japan), together with NRC (Canada), NSC and ASIAA (Taiwan), and KASI (Republic of Korea), in cooperation with the Republic of Chile. The Joint ALMA Observatory is operated by ESO, AUI/NRAO and NAOJ. The National Radio Astronomy Observatory is a facility of the National Science Foundation operated under cooperative agreement by Associated Universities, Inc. This work was supported by the Programme National "Physique et Chimie du Milieu Interstellaire" (PCMI) of CNRS/INSU with INC/INP co-funded by CEA and CNES.


Alejandro E Montecinos

Oscar R Huanel

María E Ramírez

Marie-Laure Guillemin
email: marielaure.guillemin@gmail.com

MOLECULAR DATA REVEAL THE PRESENCE OF THREE PLOCAMIUM SPECIES WITH COMPLEX PATTERNS OF DISTRIBUTION IN SOUTHERN CHILE

Keywords: Species delimitation, genetic species, long-distance dispersal, speciation, Rhodophyta, red algae

ABSTRACT

Plocamium is a widespread genus for which forty-five species are currently recognized. However, classical taxonomy, based only on morphological characters, is problematic within this genus. The use of molecular tools has uncovered cryptic genetic species, mistakenly grouped under the name of morphological species that are common and widespread (including the generitype Plocamium cartilagineum). The aim of this work was to evaluate the species diversity of Plocamium in southern Chile. For this purpose, three independent molecular markers were sequenced in samples collected from seven populations located between 41°S and 54°S. The species diversity was evaluated using phylogenetic reconstructions and two independent methods for species delimitation (ABGD and GMYC). The outcomes of each method were congruent, suggesting the presence of three species in southern Chile. One species, named Plocamium sp. 1, is restricted to Punta Guabún, the only locality sampled north of the biogeographic barrier of the 42°S. The other two species, Plocamium sp. 2 and 3 are distributed in sympatry in Patagonia and Tierra del Fuego. The three Chilean species form a clade phylogenetically close to sequences obtained from New Zealand and Australia and a divergence along the coasts of Chile after past transoceanic dispersal is proposed. We propose that divergence in glacial microrefugia could have subsequently happen in the southern part of the coast, this hypothesis being supported by the strong impact of glacial maxima on population dynamics, especially in Plocamium sp.

INTRODUCTION

Plocamium Lamouroux is a cosmopolitan genus of red seaweed for which 45 species have been recognized to the date [START_REF] Guiry | [END_REF]). These species have been recorded from the Arctic to the Antarctic, in intertidal and subtidal waters [START_REF] Wynne | A description of Plocamium fimbriatum sp. nov. (Plocamiales, Rhodophyta) from the Sultanate of Oman, with a census of currently recognized species in the genus[END_REF]. This genus has recently received more attention due its relevance in the production of bioproducts [START_REF] Calegario | Environmental modulation of the proteomic profiles from closely phylogenetically related populations of the red seaweed Plocamium brasiliense[END_REF], including anticancer molecules [START_REF] Antunes | Identification and in vitro anti-esophageal cancer activity of a series of halogenated monoterpenes isolated from the South African seaweeds Plocamium suhrii and Plocamium cornutum[END_REF][START_REF] Alves | Algae from Portuguese coast presented high cytotoxicity and antiproliferative effects on an in vitro model of human colorectal cancer[END_REF], herbicides [START_REF] Gressler | -Biochemical composition of two red seaweed species grown on the Brazilian coast[END_REF][START_REF] Pereira R | Chemical defense in the red seaweed Plocamium brasiliense: spatial variability and differential action on herbivores[END_REF] and molecules with anti-herbivore properties [START_REF] San- | -Insecticide and acaricide activities of polyhalogenated monoterpenes from Chilean Plocamium cartilagineum[END_REF][START_REF] Pereira R | Bioprospecting for bioactives from seaweeds: potential, obstacles and alternatives[END_REF]. Thus, the development of molecular studies allowing clear species recognition and a better understanding of the evolutionary history of this genus, are critical for subsequent research on these biomolecules of potential importance.

Traditional taxonomy of Plocamium species is based on the number of ramuli (i.e., small protrusions occurring along the thalli main or secondary axes) in alternating series, the width, colour, length, consistency of the thallus, the morphology of the lower ramulus, the arrangement of tetrasporangial structures and cystocarps [START_REF] Simons R | Species of Plocamium on the South African Coast[END_REF][START_REF] Womersley | The genus Plocamium (Rhodophyta) in southern Australia[END_REF][START_REF] South | -A revision of the genus Plocamium Lamouroux (Rhodophyta, Gigartinales) in New Zealand[END_REF][START_REF] Gabrielson | The marine algae of British Columbia, northern Washington and Southeast Alaska: division Rhodophyta (red algae), class Rhodophyceae, order Gigartinales, families Caulacanthaceae and Plocamiaceae[END_REF]Cremades et al. 2011). However, these morphological characters have been recognized as insufficient to describe and distinguish between some species [START_REF] Yano | Morphological homoplasy in Japanese Plocamium species (Plocamiales, Rhodophyta) inferred from the Rubisco spacer sequence and intracellular acidity[END_REF][START_REF] Saunders | Molecular divergence and morphological diversity among four cryptic species of Plocamium (Plocamiales, Florideophyceae) in northern Europe[END_REF]Cremades et al. 2011). For example, the generitype species, Plocamium cartilagineum (Linnaeus) P.S.Dixon is purportedly very widespread, being recorded in the North Atlantic, eastern and western North Pacific, northern Arabian Sea, Australia, New Zealand, Antarctica and Chile (Bischoff-Basmann & Wiencke 1996;[START_REF] Wynne | A description of Plocamium fimbriatum sp. nov. (Plocamiales, Rhodophyta) from the Sultanate of Oman, with a census of currently recognized species in the genus[END_REF]. However, molecular studies have demonstrated that specimens named as P. cartilagineum based on morphological characters actually represented various cryptic species that could be easily distinguished genetically. For example, in a study using molecular nuclear marker LSU sequences for numerous morphological specimens of P. cartilagineum from northern Europe, four genetic cryptic species were revealed [START_REF] Saunders | Molecular divergence and morphological diversity among four cryptic species of Plocamium (Plocamiales, Florideophyceae) in northern Europe[END_REF]. Moreover, another case of cryptic genetic species has been reported using the molecular marker [START_REF] Tellier | Phylogeographic analyses of the 30°S south-east Pacific biogeographic transition zone establish the occurrence of a sharp genetic discontinuity in the kelp Lessonia nigrescens: Vicariance or parapatry?[END_REF][START_REF] Montecinos | Species replacement along a linear coastal habitat: phylogeography and speciation in the red alga Mazzaella laminarioides along the south east pacific[END_REF][START_REF] Haye | Phylogeographic structure in benthic marine invertebrates of the southeast Pacific coast of Chile with differing dispersal potential[END_REF]. These transition zones have been related to the effect of historical processes, mostly linked to eustatic or climatic changes associated with Pleistocene glacial cycles [START_REF] Avise | Phylogeography: the history and formation of species[END_REF]. In the case of Plocamium, various widespread morphological species, encompassing more than one biogeographic area, have been reported. In these taxa, the use of molecular markers and genetic species delimitation approaches can help in reevaluating species diversity and distribution.

The Chilean coast is subdivided in three major biogeographical regions (Camus 2001): the Peruvian Province (PP), located from Peru to a southern limit around 30-33°S on the northern coast of Chile; the Magellanic Province (MP) extending from Cape Horn (56°S) north to 41-42°S (Chiloé Island) and an Intermediate Area (IA) limited by the PP and MP provinces. These biogeographical regions are characterized by distinct biota from warm-temperate in PP to cold-water and sub-Antarctic species in MP. The IA is characterized by a gradual overlap of biota characteristic of the other two provinces [START_REF] Camus P | -Biogeografía marina de Chile continental[END_REF]. Phylogeographic breaks concordant with the biogeographic limit at 41°S -42°S have been reported for various marine or coastal species, such as Acanthina monodon Pallas, a brooding gastropod, [START_REF] Sánchez R | Spatial pattern of genetic and morphological diversity in the direct developer Acanthina monodon (Gastropoda: Mollusca)[END_REF]) and the Patagonian otter Lontra provocax Thomas [START_REF] Vianna | Phylogeography of the Patagonian otter Lontra provocax: adaptive divergence to marine habitat or signature of southern glacial refugia?[END_REF]. The coastline north of 42°S is continuous, linear and dominated by rocky shores only intersected by a few small rivers and sandy beaches, while a high density of islands, fjords and channels, influenced by sub-Antarctic oceanographic and climatic conditions, characterizes the shoreline south of 42°S. These distinct coastal morphologies are the results of major topographic transformations due to interglacial/glacial cycles during the Pliocene and Pleistocene in southern Chile [START_REF] Mercer | Glacial history of Southernmost South America[END_REF][START_REF] Mcculloch R | Climatic inferences from glacial and palaeoecological evidence at the last glacial termination, southern South America[END_REF].

During glacial maxima, in particular during the Last Glacial Maximum (LGM, 20,000 years ago), ice sheets covered a broad region of southern Chile from the Chiloé Island at 41°S to Cape Horn at 56°S [START_REF] Hulton | The Last Glacial Maximum and deglaciation in southern South America[END_REF][START_REF] Saillard | Non-steady long-term uplift rates and Pleistocene marine terrace development along the Andean margin of Chile (31°S) inferred from 10 Be dating[END_REF]. Glacial periods were characterized by the retraction of the temperate biota in glacial refugia while interglacial periods were characterized by range expansion of these species in areas previously covered by ice.

Along the Chilean coast, three species of Plocamium identified using morphology have a distribution range that span more than one Chilean bioregion: P. cartilagineum (in the Peruvian Province, the Intermediate Area and the Magellanic Province), P. secundatum (Kützing) Kützing (in the Peruvian and Magellanic Province), and P. pacificum Kylin (in the Peruvian Province and Intermediate Area) [START_REF] Etcheverry | Algas marinas bentónicas de Chile[END_REF][START_REF] Ramírez | Catálogo de algas marinas bentónicas de la costa templada del Pacífico de Sudamérica[END_REF][START_REF] Ramírez | Flora marina bentónica de la región austral de Sudamérica y la Antártica[END_REF]). In addition to the extensive distribution of these morphological species, cryptic species have already been reported in P. cartilagineum [START_REF] Saunders | Molecular divergence and morphological diversity among four cryptic species of Plocamium (Plocamiales, Florideophyceae) in northern Europe[END_REF]Cremades et al. 2011), casting doubts about Plocamium diversity and distribution in Chile. The present work aims to explore Plocamium species diversity and evolutionary history in southern Chile (41°S-54°S) using genetic markers encompassing the three cell compartments (i.e., mitochondria, chloroplast and nucleus). Using these molecular data sets, we applied different methods to delimit species (as conceptualized by the phylogenetic species concept; de Queiroz 1998) and improve knowledge on Plocamium diversity. We hypothesized that genetic species, if existent, will show distributions limited by the biogeographic barrier at 42°S and that species encountered in Patagonia and Tierra del Fuego will present historical population dynamics (i.e., contraction during the LGM and recent expansion) strongly affected by the glacial/interglacial cycles.

MATERIALS AND METHODS

Sampling

A total of 107 individuals of Plocamium spp. were collected from 7 localities (Table 1). One locality was located north of the 42°S (i.e., Punta Guabún) and six localities were located south of the 42°S (i.e., San Gregorio, Parque Chabunco, Faro Porvenir, Los Canelos, Faro San Isidro and Fiordo Yendegaia). Intertidal samplings were conducted during diurnal low tide in Los Canelos and Fiordo Yendegaia while subtidal samplings were done by means of SCUBA diving in the rest of the localities studied. Each sample corresponds to a single frond cut from an isolated holdfast.

In the field, all the samples were first named 'P. cartilagineum'. However, more precise observations in the lab show that some individuals from the southern part of the country presented a ramification pattern not fully congruent with the one expected for P. cartilagineum (slender ramuli, not all ramification unilateral, very bushy in appearance). In the same way, samples from Punta Guabún did not present the typical P. cartilagineum morphology. These small plants were characterized by an intense red color with sympodial branching characterized by profuse ramifications almost from the base and third and fourth order branches arranged in a scalloped manner. Because of these slightly distinct morphologies and since only a few mature tetrasporophytes were sampled we choose to refer to all individuals studied here as Plocamium spp. For most individuals (i.e., 96) only small tissue fragments were conserved in silica and no observation in the lab could be made. The 11 samples guarded as voucher specimens are housed in the herbarium of the National Museum of Natural History, Chile (SGO, see voucher numbers in Supplementary Table 1) DNA extraction, PCR amplification, sequencing and sequence alignment Dry tissues were ground by hand in liquid nitrogen and DNA was extracted using extraction kit E.Z.N.A.® Poly-Gel DNA Extraction (Omega Bio-Tek Inc., Norcross, USA). Three independent genetic markers were used to provide complementary species identification based on molecular criterion: a partial sequence of the mitochondrial Cytochrome c Oxidase I gene (5P-COI); a partial sequence of the plastid gene rbcL, encoding the large subunit of the ribulose-1,5-bisphosphate (rbcL) and the partial large subunit ribosomal RNA gene (LSU). The amplification of 5P-COI was performed, for all specimens sampled, using the primers developed by [START_REF] Saunders | Applying DNA barcoding to red macroalgae: a preliminary appraisal holds promise for future applications[END_REF]. The amplification of the rbcL marker was performed for a subset of samples (n = 18) using the primers developed by [START_REF] Hommersand | Phylogenetic systematics and biogeography of the Gigartinaceae (Gigartinales Rhodophyta) based on sequence-analysis of rbcL[END_REF]. The LSU was amplified as three overlapping fragments with previously published primer combinations [START_REF] Harper | The application of sequences of the ribosomal cistron to the systematics and classification of the florideophyte red algae (Florideophyceae Rhodophyta)[END_REF], in a subset of five samples. For the 5P-COI, PCR were performed using conditions described in Dubrasquet et al. (2018) while rbcL and LSU markers were amplified using conditions described in [START_REF] Hommersand | Phylogenetic systematics and biogeography of the Gigartinaceae (Gigartinales Rhodophyta) based on sequence-analysis of rbcL[END_REF] and [START_REF] Harper | The application of sequences of the ribosomal cistron to the systematics and classification of the florideophyte red algae (Florideophyceae Rhodophyta)[END_REF], respectively. For both rbcL and LSU genes, sub samples included individuals from the three genetic groups recovered by the 5P-COI (more details in the results section below). PCR products were purified using commercial kit E.Z.N.A.® DNAProbe Purification (Omega Bio-Tek Inc., Norcross, USA) and sequenced with primers used for amplification at the AUSTRAL-omics Core-Facilities (Valdivia, Chile). Sequences were aligned manually using Mega X [START_REF] Kumar | MEGA X: Molecular Evolutionary Genetics Analysis across Computing Platforms[END_REF]) and checked by eye; only traces with high quality values and no ambiguities were retained for further analyses.

Phylogenetic analyses

To explore phylogenetic relationships between Plocamium species, 250 5P-COI sequences from the genus available in GenBank were added to our data set. In the same way, 24 and 20 sequences from GenBank were added to the rbcL and LSU data sets, respectively. Phylogenetic analyses were conducted independently for each gene, using both Maximum Likelihood (ML) and Bayesian Inference (BI) methods. ML analyses were performed using W-IQ-Tree [START_REF] Trifinopoulos | W-IQ-TREE: a fast online phylogenetic tool for maximum likelihood analysis[END_REF]. The best-fit substitution model was selected using the Bayesian Information Criterion [START_REF] Kalyaanamoorthy | ModelFinder: fast model selection for accurate phylogenetic estimates[END_REF] implemented in W-IQ-Tree. The selected model was K3Pu+F+I+G4 for the 5P-COI, TN+F+I+G4 for the rbcL and GTR+F+I+G4 for the LSU. BI analyses were conducted using MrBayes v3.2.7 [START_REF] Ronquist | MRBAYES 3.2: Efficient Bayesian phylogenetic inference and model selection across a large model space[END_REF]. Two independent analyses were run using, for each one, four chains and 20 million generations. Trees and parameters were sampled every 1,000 generations and the default parameters for temperature and branch swapping were used. The first 20% of the sampled trees were discarded as "burn-in" to ensure stabilization. The remaining trees were used to compute a consensus topology and posterior probability values.

The split frequency (variance among the four independent runs) was below 0.005, confirming that the posterior probability distribution was accurately sampled.

Delimitation of genetic species

To evaluate the existence of genetic species, two independent analyses were conducted using the 5P-COI dataset. First, the Automatic Barcode Gap Discovery (ABGD) was remotely run at https://bioinfo.mnhn.fr/abi/public/abgd/abgdweb.html. ABGD identifies a value separating the frequency distribution of intraspecific pairwise genetic distances from the ones of interspecific pairwise genetic distances, even when they overlap, and use it as a threshold to delimit species [START_REF] Puillandre | ABGD, Automatic Barcode Gap Discovery for primary species delimitation[END_REF]. We computed Kimura two-parameter (K2P) genetic distances and used default ABGD settings.

Second, a General Mixed Yule Coalescent (GMYC) analysis was run. GMYC identifies a threshold value for the shift in branching rate from coalescent lineage branching to interspecific diversification on an ultrametric tree and explicitly delimits "independently evolving" clusters (i.e., putative species; [START_REF] Pons | Sequence-based species delimitation for the DNA taxonomy of undescribed insects[END_REF][START_REF] Monaghan | Accelerated species inventory on Madagascar using coalescent-based models of species delineation[END_REF]. Before the analysis, duplicated haplotypes were removed from the data set using DnaSP v6.12.03 [START_REF] Rozas | -DnaSP v6: DNA Sequence Polymorphism Analysis of Large Datasets[END_REF]. Branch lengths were estimated under a relaxed log-normal clock using the Bayesian analysis implemented in BEAST v1.10.4 [START_REF] Suchard | Bayesian phylogenetic and phylodynamic data integration using BEAST 1.10[END_REF]. A coalescent (constant size) prior was used and Markov Chains Monte Carlo (MCMC) were run for 20 million generations. Trees were sampled each 1,000 generations with a 10% burn-in. A visual inspection of MCMC progression using Tracer v1.7.1 [START_REF] Rambaut | Posterior summarisation in Bayesian phylogenetics using Tracer 1.7[END_REF]) was performed to corroborate stabilization. An ultrametric tree was constructed using Tree Annotator v1.10.4 (Rambaut & Drummond 2018). Since the multiple-thresholds approach tends to overestimate the number of delineated species [START_REF] Fujisawa | Delimiting species using singlelocus data and the Generalized Mixed Yule Coalescent (GMYC) approach: A revised method and evaluation on simulated datasets[END_REF] only the single-threshold [START_REF] Pons | Sequence-based species delimitation for the DNA taxonomy of undescribed insects[END_REF] versions of GMYC was fitted on the ultrametric tree using the SPLITS v1.0-19 package for R (https://r-forge.r-project.org/projects/splits/).

Genetic diversity, network reconstruction and historical demography

Within each genetic species, defined using ABGD and GMYC, four diversity indices were calculated for the 5P-COI gene using DnaSP v6.12.03 [START_REF] Rozas | -DnaSP v6: DNA Sequence Polymorphism Analysis of Large Datasets[END_REF]): the number of haplotypes (nH), the number of polymorphic sites (S), gene diversity (H, [START_REF] Nei | Molecular Evolutionary Genetics[END_REF]) and nucleotide diversity (π, [START_REF] Nei | Mathematical model for studying genetic variation in terms of restriction endonucleases[END_REF]. Moreover, within each genetic species, haplotype networks were reconstructed for the 5P-COI using the median-joining algorithm implemented in NETWORK v10.1.0.0 [START_REF] Bandelt | Median-Joining networks for inferring intraspecific phylogenies[END_REF].

Finally, to evaluate changes in the demographic history of the genetic species, two complementary approaches were used to infer the historical demography using the 5P-COI dataset. First, Tajima's D [START_REF] Tajima | Statistical method for testing the neutral mutation hypothesis by DNA polymorphism[END_REF]) and Fu's Fs [START_REF]Statistical tests of neutrality of mutations against population growth, hitchhiking and background selection[END_REF] statistics were calculated to detect significant past changes in population size.

Significant departure from selection-drift equilibrium was tested using 1,000 bootstrap replicates in Arlequin v3.5.2.2 (Excoffier & Lischer 2010). Under the presumption of neutrality, negative values distinguish populations in expansion while positive values, associated to the loss of rare alleles, are considered as signature of recent bottleneck [START_REF] Tajima | Statistical method for testing the neutral mutation hypothesis by DNA polymorphism[END_REF][START_REF]Statistical tests of neutrality of mutations against population growth, hitchhiking and background selection[END_REF]

RESULTS

A total of 107 sequences of Plocamium spp. collected from 7 localities were obtained for the 5P-COI (575bp; Table 1). Moreover, a sub sample of 18 and 5 individuals were sequenced for the rbcL (641bp) and the LSU (2960bp), respectively (Table 1). GenBank accession numbers for the three molecular markers sequenced are available in the Supplementary Table 1.

Phylogenetic relationships

Phylogenetic relationships based on 5P-COI, for both the ML and BI analyses, recovered all specimens sequenced in the present study as a single, wellsupported monophyletic group, strongly divergent from all the other sequences of Plocamium available in GenBank (Fig. 1). This Chilean clade appear as nested within a poorly resolved group including most specimens sampled in Australia and New Zealand, and as sister to a well-supported monophyletic group composed by specimens of P. cartilagineum from New Zealand, P. patagiatum J.Agardh from Australia and P. angustum from Australia and New Zealand (Fig. 1). It is interesting to note that specimens sampled within the same ocean tend to be genetically related (Fig. 1). The Chilean clade was also recovered as sister of South Hemisphere Plocamium species for the rbcL (i.e., as sister to KC174809, U21703, U26821 and HQ224543 from New Zealand, Fig. S1) and for the LSU (as sister to AY881712 and AY881714 from Australia, Fig. S2). Finally, Chilean sequences from the present study form three monophyletic lineages observed for the three genetic markers used (Fig. 1, Fig. S1, Fig. S2). Whatever the gene under study, these lineages were generally well supported for both the ML and BI analyses (Fig. 1, Fig. S1, Fig. S2).

Species delimitation

Genetic pairwise K2P distances for the 5P-COI ranged from 0 to 0.062 and the ABGD located the barcode gap within the 0.010-0.030 distance range (Fig. S3b).

Primary partitions using this threshold suggested the existence of three genetic groups (Fig. 2, Fig. S3a). The likelihood of the GMYC model for the single threshold model was (LGMYCsingle = 44.04); a value significantly higher than the one obtained for the null model (L0 = 40.86). The number of partitions obtained for the GMYC was three, with confidence limits of three to five (Fig. 2, Fig. S4). The three monophyletic groups recovered using phylogenetic reconstructions (Fig. 1, S1 and S2) were supported as putative species by both the ABGD and the GMYC single-threshold results (Fig. 2). Moreover, values of Kimura 2-parameter (K2P) were more than ten times higher when measured between genetic species (Plocamium sp. Gregorio, Faro Porvenir and Faro San Isidro) (Fig. 3). The 19 samples from Parque Chabunco were identified as Plocamium sp. 3.

Genetic diversity, haplotype network and demographic history.

For the 5P-COI data set the number of haplotypes (nH) and number of polymorphic sites (S) were the lowest in Plocamium sp. 1 (nH = 2; S = 1) and the highest in Plocamium sp. 3 (nH = 6; S = 6; Table 2). The highest values of genetic and nucleotide diversity were encountered in Plocamium sp. 2 (H = 0.684 ± 0.099 and π = 0.00144 ± 0.00111), while the lowest values were encountered in Plocamium sp. 1 (H = 0.118 ± 0.101 and π = 0.00019 ± 0.00048) (Table 2).

Plocamium sp. 2 presented a haplotype network slightly more reticulated than the two other species, for which star-like type of networks were observed (Fig. 3). In the haplotype network Plocamium sp. 2 was connected to Plocamium sp. 3 by 25 mutational steps, while Plocamium sp. 1 was connected to Plocamium sp. 2 by 18 mutational steps. Whatever the species under study, all values of Tajima's D and Fu's Fs statistics were negative, but these were significant only for Plocamium sp. 3 (Table 3). Mismatch distributions were unimodal with the most commonly calculated number of differences between pairs of sequences equal to 0 in Plocamium sp. 1 and Plocamium sp. 3 and to 1 in Plocamium sp. 2 (Fig. 4). Tests for goodness-of-fit based on the sum of square deviations (SSD) for the demographic expansion model give values ranking from 0.00012 (p-value = 0.38400) for Plocamium sp. 1 and 0.00028 (p-value = 0.34400) for Plocamium sp. 3 up to 0.01945 (p-value = 0.23400) for Plocamium sp. 2 (Table 3). These results did not reject the null hypothesis of a population expansion in any of the three genetic species studied. especially in taxa with high capacity of dispersal, for example fish [START_REF] Blower | Population genetics of Australian white sharks reveals fine-scale spatial structure, transoceanic dispersal events and low effective population sizes[END_REF] or crustacea [START_REF] Page | Historical transoceanic dispersal of a freshwater shrimp: the colonisation of the South Pacific by the genus Paratya (Atyidae)[END_REF]. Recurrent dispersal, more than vicariance, has indeed been postulated to be the mechanism leading to the biogeographic patterns and disjunct species distributions observed nowadays in the Southern Ocean [START_REF] Waters | Driven by the West Wind Drift? A synthesis of southern temperate marine biogeography, with new directions for dispersalism[END_REF][START_REF] Fraser | Genetic affinities between transoceanic populations of non-buoyant macroalgae in the high latitudes of the Southern Hemisphere[END_REF]. The importance of transoceanic rafting is less recognized in marine species with limited dispersal capacity (i.e., lack of larvae or short lived propagules) even if recent studies have demonstrated that this mechanism, depending on the species physiological/reproductive tolerance, could be highly efficient, allowing rapid expansion of their distribution ranges (Thiel & Gutow 2005a;2005b;[START_REF] Fraser | -Oceanic rafting by a coastal community[END_REF][START_REF] Fraser | Genetic affinities between transoceanic populations of non-buoyant macroalgae in the high latitudes of the Southern Hemisphere[END_REF]Waters et al. 2013;[START_REF] Valero | Tracing the trans-pacific evolutionary history of a domesticated seaweed (Gracilaria chilensis) with archaeological and genetic data[END_REF][START_REF] Valero | Phylogeography of seaweeds in the South East Pacific: Complex evolutionary processes along a latitudinal gradient[END_REF][START_REF] Tala | Long-term persistence of the floating bull kelp Durvillaea antarctica from the South-East Pacific: Potential contribution to local and transoceanic connectivity[END_REF]). In the Southern Hemisphere, currents are dominated by the Antarctic Circumpolar Current (ACC) and the West Wind Drift (WWD) [START_REF] Waters | Driven by the West Wind Drift? A synthesis of southern temperate marine biogeography, with new directions for dispersalism[END_REF]. Recurrent dispersal from Australia and/or New Zealand to Chile have been registered using molecular data in various macroalgae including the buoyant seaweed Durvillaea antarctica (Chamisso) Hariot [START_REF] Fraser | -Kelp genes reveal effects of subantarctic sea ice during the Last Glacial Maximum[END_REF]), but also the non-buoyant species Bostrychia intricata (Bory) Montagne, Adenocystis utricularis (Bory)

DISCUSSION

Skottsberg [START_REF] Fraser | Genetic affinities between transoceanic populations of non-buoyant macroalgae in the high latitudes of the Southern Hemisphere[END_REF] New Zealand to temperate Chile that was followed by genetic divergence leading to the speciation of D. incurvata (Suhr) Macaya (a species restricted to Chilean temperate waters) some 3 -10 Million years ago [START_REF] Fraser | Genetic affinities between transoceanic populations of non-buoyant macroalgae in the high latitudes of the Southern Hemisphere[END_REF][START_REF] Fraser | The biogeographic importance of buoyancy in macroalgae: A case study of the southern bull kelp genus Durvillaea (Phaeophyceae), including descriptions of two new species[END_REF]. Studies in other organisms, as in the coastal sac spiders of the genus Amaurobioides O. Pickard-Cambridge, show repeated events of long-distance dispersal along the WWD followed by divergence, revealing a remarkable pattern of "stepping-stone" speciation all around the Southern Ocean (Ceccarelli et al. Contrasting with Plocamium sp. 1, the species Plocamium sp. 2 and 3 were in sympatry in most localities sampled in Patagonia and Tierra del Fuego. Existence of cryptic genetic species or diverged haplotypic groups distributed in sympatry in southern Chile have already been reported in three macroalgae: Adenocystis utricularis, Bostrychia intricata [START_REF] Fraser | Genetic affinities between transoceanic populations of non-buoyant macroalgae in the high latitudes of the Southern Hemisphere[END_REF] and Iridaea cordata (Turner)

2016).

Speciation in the genus

Bory [START_REF] Ocaranza-Barrera P | Molecular divergence between Iridaea cordata (Turner) Bory de Saint-Vincent from Antarctic Peninsula and the Magellan Region[END_REF]. In these cases, divergence in sympatry or microallopatry could be hypothesized. Exhaustive surveys have revealed common patterns of genetic divergence, consistent with isolation in refugia during glacial periods, in various organisms [START_REF] Hewitt | Genetic consequences of climatic oscillations in the Quaternary[END_REF][START_REF] Sérsic | Emerging phylogeographic patterns of plants and terrestrial vertebrates from Patagonia[END_REF]

  3. DES DONNÉES MOLÉCULAIRES RÉVÈLENT LA PRÉSENCE DE TROIS ESPÈCES DE PLOCAMIUM PRESENTANT UN PATRON COMPLEX DE DISTRIBUTION DANS LE SUD DU CHILI RESUMÉ Quarante-cinq espèces sont actuellement reconnues dans le genre Plocamium, un genre présentant une très ample distribution. Cependant, la taxonomie classique, basée uniquement sur les caractères morphologiques, est problématique au sein de ce genre. L'utilisation d'outils moléculaires a permis de révéler l'existence d'espèces génétiques cryptiques, groupées par erreur sous un même nom, celui d'espèces morphologiques courantes et répandues (y compris l'espèce type du genre Plocamium: Plocamium cartilagineum). Le but de notre travail était d'évaluer la diversité d'espèces de Plocamium dans le sud du Chili. A cet effet, trois marqueurs moléculaires indépendants ont été séquencés pour des échantillons prélevés dans sept populations situées entre 41°S et 54°S. La diversité en terme d'espèces a été évaluée à l'aide de reconstructions phylogénétiques et de deux méthodes indépendantes de délimitation d'espèces génétiques (ABGD et GMYC). Les résultats des différentes méthodes sont congruents, suggérant la présence de trois espèces dans le sud du Chili. Une espèce, nommée Plocamium sp. 1, est limité à Punta Guabún, la seule localité échantillonnée au nord de la barrière biogéographique du 42°S. Les deux autres espèces, Plocamium sp. 2 et 3 sont distribuées en sympatrie en Patagonie et en Terre de Feu. Les trois espèces chiliennes forment un clade phylogénétiquement proche de séquences obtenues en Nouvelle-Zélande et en Australie et une divergence le long des côtes du Chili après un évènement historique de dispersion transocéanique passée pourrait expliquer ce résultat. Nous proposons des phénomènes postérieurs de divergence en micro-refuges glaciaires comme moteur de la spéciation en Patagonie et en Terre de Feu. Cette hypothèse est étayée par le fort impact des maxima glaciaires sur la dynamique des populations, en particulier dans le cas de Plocamium sp. 3. Mots clés: Délimitation d'espèces, espèces génétiques, dispersion à longue distance, spéciation, Rhodophyta, algues rouges.

  5P-COI, in individuals of P. angustum (J.Agardh) J.D.Hooker & Harvey from Australia and New Zealand (Cremades et al. 2011). Contrasting with P. cartilagineum and P. angustum, some taxonomically recognized species, as for example P. nanum G.W.Saunders & Lehmkuhl, show restricted distributions (Saunders & Lehmkuhl 2005) probably linked to the presence of biogeographical barriers. Biogeographical barriers are zones defined by rapid changes in biota that can act as barriers to migration (Dawson 2001). In the marine realm, biogeographical barriers have often been associated with landscape features, such as the presence of strong currents or topographical features (e.g., sandy beaches, river mouth, sea mount) limiting gene flow between populations and have been reported as important drivers of speciation, especially in taxa presenting low dispersal capacity (Avise 2000; Kuo & Avise 2005). Deep phylogeographic discontinuities, congruent with biogeographic barriers, have commonly been encountered in widespread species with distributions encompassing various biogeographic areas (Dawson 2001; Hurt et al. 2009). For example, numerous studies have uncovered deep genetic divergence in coastal marine taxa that coincide with recognized transition zones, such as the California transition zone (Dawson 2001; Kelly & Palumbi 2010) and the 30°S-33°S area located along the Chilean coast

  . Second, the observed mismatch distributions of the number of differences between pairs of 5P-COI sequences were compared to estimated values under a model of demographic expansion (Roger & Harpending 1992) using Arlequin v3.5.2.2 (Excoffier & Lischer 2010). Multimodal distributions generally characterize populations in demographic equilibrium while unimodal distributions are associated with recent expansion.

  Our study revealed the existence of three genetic species of Plocamium in southern Chile, with Plocamium sp. 2 and Plocamium sp. 3 located in sympatry in Patagonia and Tierra del Fuego while Plocamium sp. 1 was only encountered north of the biogeographical limit at 42°S. Interestingly, phylogenetic analyses recovered the Chilean clade as sister to a well-supported monophyletic group composed by specimens from New Zealand and Australia, suggesting the occurrence of transoceanic dispersal in the past. On the other hand, various paraphyletic taxa were observed within Plocamium phylogenetic trees (e.g., P. cartilagineum, P. patagiatum, P. angustum, P. fimbriatum M.J.Wynne, P. violaceum and P. pacificum; Fig. 1), clearly pointing out the difficulty of species identification based on morphological characters in this genus (Cremades et al. 2011). Concordance across results obtained with different methods (here GMYC and ABGD) and monophyly recovered in trees reconstructed with unlinked markers are now widely acknowledged as suitable for genetic species delimitation (Carstens et al. 2013; Modica et al. 2014). Our results confirm the relevance of information obtained from molecular markers encompassing the three cell compartments (i.e., mitochondria, chloroplast and nucleus) to delimit species in the genus Plocamium and better estimate species diversity, distribution and understand the evolutionary history in these highly morphologically variable red algae. Transoceanic dispersal as potential origin of Chilean Plocamium species ancestral clade In the phylogenetic trees, the three Chilean Plocamium species formed a clade embedded in sequences from the Southern Hemisphere and genetically close to clades from New Zealand and Australia. We propose that the Chilean Plocamium clade has diverged from Australian or New Zealand colonists, after their arrival by rafting in Chile. Transoceanic dispersal has commonly been reported,

,

  Capreolia implexa Guiry & Womersley (Boo et al. 2014) and Agarophyton chilense (C.J.Bird, McLachlan & E.C.Oliveira) Gurgel, J.N.Norris et Fredericq (as Gracilaria chilensis C.J.Bird, McLachlan & E.C.Oliveira; Guillemin et al. 2014). All these species show genetic signatures of recent west to east dispersal across vast oceanic distances. In the case of the genus Durvillaea Bory there is evidence of a long-distance dispersal event from

  Plocamium along the coast of southern Chile After transpacific colonization, distinct processes of divergence seem to have taken place during the radiation of the genetic Chilean Plocamium species ancestral clade. The species Plocamium sp. 1 is found isolated north of the biogeographical limit of 42°S and we propose that parapatric or allopatric speciation could have taken place in this case. A strong biogeographic discontinuity has been described at 41-42°S (Camus 2001), generally related to the latitudinal migration of the southern Westerlies during the Miocene-Pleistocene. The split of the WWD into the northward Humboldt Current and the southward Cape Horn Current, located at these latitudes, has been demonstrated to represent a major oceanic barrier that has contributed to the origin of the biogeographic break. Nowadays, contrasted ecologic, climatic and topographic features characterize both sides of the 41-42°S biogeographical limit (Camus 2001). In the past, major currents restricting gene flow could have led to the divergence of Plocamium sp. 1 from Plocamium sp. 2 and 3; while more subtle differences in term of coastal topography or salinity could also help in maintaining these species genetic integrity nowadays. Our sampling does not allow separation of patterns of allopatric from parapatric speciation nor to precisely pinpoint the phylogeographic break in Plocamium (i.e., the gap between IA and MP sampling sites span more than 1,200 kilometers). However, other genetic studies described a phylogeographic break at the 41°S-42°S zone or nearby (the buoyant kelps Durvillaea spp., Fraser et al. 2010; in the brooding gastropod, Acanthina monodon, Sánchez et al. 2011; the Patagonian otter Lontra provocax, Vianna et al. 2011). Theoretical studies emphasize the possible quick genetic divergence that could be observed in scenarios of parapatric speciation in organisms with low dispersal capacity, as is the case for Plocamium, (Gravilets et al. 2000; Kuo & Avise 2005). Moreover, parapatric speciation has been suggested as a common mechanism of speciation in macroalgae along the Chilean coast (brown algae: Lessonia trabeculata Villouta & Santelices and L. spicata (Suhr) Santelices, as L. nigrescens Bory in Tellier et al. 2009; red alga: Mazzaella laminarioides (Bory) Fredericq, Montecinos et al. 2012).

Figure 1 -

 1 Figure 1 -Maximum likelihood (ML) phylogram of the genus Plocamium based

  Figure S1 -Maximum likelihood (ML) phylogram of the genus Plocamium based on rbcL sequences. ML bootstrap (BS)/Bayesian posterior probability (PP) values are shown above or close to each branch and only values superior to 75 and 0.75, respectively, are given. Colors correspond to oceans where individuals sequenced where sampled. Outgroup corresponds to Sarcodia ciliata (GenBank accession: KM360040).

Figure S2 -Figure S3 -

 S2S3 Figure S2 -Maximum likelihood (ML) phylogram of the genus Plocamium based on LSU sequences. ML bootstrap (BS)/Bayesian posterior probability (PP) values are shown above or close to each branch and only values superior to 75 and 0.75, respectively, are given. Colors correspond to oceans where individuals sequenced where sampled. Outgroup corresponds to Sarcodia ciliata (GenBank accession: DQ343708).

  Plocamium sp. 2 and Plocamium sp. 3. Plocamium sp. 1 was restricted to Punta Guabún, the only locality sampled north of the 42ºS in the present study, while the two other species were distributed in sympatry in southern Chile (Fig.3). The two species

	Plocamium sp. 2 and Plocamium sp. 3 were collected at the same sites in both
	intertidal (i.e., Los Canelos and Fiordo Yendegaia) and subtidal (i.e., San
	1 -Plocamium sp. 2 = 0.05361 ±
	0.00936; Plocamium sp. 1 -Plocamium sp. 3 = 0.03063 ± 0.00684; Plocamium
	sp. 2 -Plocamium sp. 3 = 0.04652 ± 0.00865) than between haplotypes
	sequenced within a single genetic species (within Plocamium sp. 1 = 0.00019 ±

0.00019; within Plocamium sp. 2 = 0.00040 ± 0.00014; within Plocamium sp. 3 = 0.00144 ± 0.00079). The three putative genetic species of Plocamium from southern Chile were then named Plocamium sp. 1,

Table 1 -

 1 . During the Pleistocene glacial/interglacial cycles populations of temperate species could have survived in isolated microrefugia in Patagonia and Tierra del Fuego (for example, terrestrial organisms: Sérsic et al. 2011; freshwater fish: Zemlak et al. 2010). In the region, during the glacial maxima, Plocamium populations could Strong ice impact during the LGM and recent demographic expansion has also been observed in other Patagonian macroalgae as Mazzaella laminarioides (Montecinos et al. 2012), Gigartina skottsbergii Setchell & N.L.Gardner (Billard et al. 2015) and Durvillaea antarctica (Fraser et al. 2009). Iridaea cordata (Ocaranza et al. 2019) and Gigartina skottsbergii (Billard et al. 2015), where cryptic sister species were encountered on both side of the Drake Passage. The main difference between Plocamium and Iridaea cordata and Gigartina skottsbergii is that Antarctic and Chilean species are not sister species in the case of Plocamium. Complex patterns of long distance colonization Iridaea cordata and Gigartina skottsbergii: Hommersand & Fredericq 2003; Billard et al. 2015; Ocaranza et al. 2019). More efforts are needed to understand the evolutionary history of Plocamium in the Southern Hemisphere including the sub-Antarctic Islands and the coasts of the Antarctic Peninsula. Sampling localities and number of individuals sequenced for the three molecular markers used in the present study. Abbreviation (CODE) and geographic coordinates are indicated.

	have survived in small pockets of suitable habitat located at the edge of, or even
	within, glaciated areas (Rull 2009; Mosblech et al. 2011). During isolation periods
	(i.e., glacial maxima), divergence between microrefugia could be favored by drift
	and/or selection. After interglacial expansion from refugia, differentiated genetic
	groups or species (e.g., Plocamium sp. 2 and 3) could then be observed in
	sympatry in localities where secondary contact takes place (Zemlak et al. 2008;
	Zhang et al. 2008; Durand et al. 2009). Supporting the impact of
	glacial/interglacial cycles on marine Chilean species living at high latitude, our
	results suggest that both Plocamium sp. 2 and 3 have been affected by these
	cycles but that the bottleneck and demographic expansion is more recent in
	Plocamium sp. 3 (i.e., bottleneck probably linked to the LGM) than in Plocamium
	sp. 2.

DE QUEIROZ K. 1998. -The general lineage concept of species, species criteria, and the process of speciation: A conceptual unification and terminological recommendations, in Howard D. J. and Berlocher, S. H. (eds.),

Table 2 -

 2 Genetic diversity estimates for the molecular marker 5P-COI. Each

	Plocamium genetic species, as delimited by ABGD and GMYC, was treated
	separately.
	Species	N	nH	H	SD	π	SD	S
	Plocamium sp. 1 17	2	0.118	0.101	0.00019	0.00048	1
	Plocamium sp. 2 17	5	0.684	0.099	0.00144	0.00111	4
	Plocamium sp. 3 73	6	0.133	0.054	0.00026	0.00093	6
	TOTAL 107

N: number of sequences; nH: number of haplotypes; H: gene diversity; π: nucleotide diversity; S: number of polymorphic sites; SD: Standard deviation.

Table 3 -

 3 Neutrality tests calculated using the 5P-COI marker data set. Results are given separately for each Plocamium genetic species (as delimited by ABGD and GMYC).

	Species	Tajima's D p-value	Fu's Fs	p-value	SSD	p-value
	Plocamium sp. 1	-1.16387	0.14500 -0.74844 0.07800 0.00012 0.38400
	Plocamium sp. 2	-0.74003	0.26300 -1.61645 0.08000 0.01945 0.23400
	Plocamium sp. 3	-2.16969	0.00000 -7.46656 0.00000 0.00028 0.34400
	SSD: Sum of square deviations.

ACKNOWLEDGMENT

This study was supported by the INACH project RG_15-16 and the FONDAP program project no. 15150003 (FONDAP IDEAL). OH is supported by BECA DE DOCTORADO NACIONAL Grant#21120791 (CONICYT, Chile). The authors are grateful to Sylvain Faugeron and his team for sample collection in Punta Guabún, Erasmo Macaya for sample collection in Fiordo Yendegaia, Mateo Caceres and Diego Bravo for their help in the field in Patagonia and Tierra del Fuego, and Kamilla Flores Robles and Hardy Guzman for lab support. We would like to thank the two reviewers for their comments and efforts towards improving our manuscript. We also thank the IDEAL staff for assistance with infrastructure, especially in Punta Arenas.

DATA SHARING AND DATA ACCESSIBILITY

The authors confirm that all data underlying the findings are fully available without restriction. All sequences are available in GenBank (accession numbers in Supplementary Table 1).

COMPETING INTEREST

The authors declare that they have no competing interests.