PhD Christophe Boudry
email: christophe.boudry@enc-sorbonne.fr

PhD Ghislaine Chartron

Keywords:

Introduction

In the last few decades, access to scientific information has almost entirely migrated to a digital environment. This changing environment requires persistent and reliable unique identifiers to manage access to digital resources. The most common identifier on the web is the Uniform Resource Locator (URL), which provides the location of resources. URLs have many limitations.

They work properly only if the resources are not removed, and do not provide a means of associating meta information (e.g. file format) about the resource which could help to identify intellectual property rights [START_REF] Carnevale | The life and death of URLs in five biomedical informatics journals[END_REF][START_REF] Ducut | An update on Uniform Resource Locator (URL) decay in MEDLINE abstracts and measures for its mitigation[END_REF][START_REF] Park | Examining success factors in the adoption of digital object identifier systems[END_REF][START_REF] Wagner | Disappearing act: decay of uniform resource locators in health care management journals[END_REF]. In biology and medicine, the PubMed Identifier (PMID) has long been operating to identify and access articles via the PubMed database. However, the scope of PMID is limited because it only includes journals indexed in PubMed in the field of biology and medicineand is functional only in this database. The lack of similar systems in other scientific fields, and therefore the lack of normalization, resulted in the establishment of the Digital Object Identifier (DOI) in 1997 [START_REF] Paskin | The digital object identifier system: digital technology meets content management[END_REF].This system, managed by the International DOI Foundation (IDF) [START_REF] Chandrakar | Digital object identifier system: an overview[END_REF]"Digital Object Identifier System Handbook" 2016;[START_REF] Paskin | The digital object identifier system: digital technology meets content management[END_REF][START_REF] Paskin | Digital object identifier (DOI) system[END_REF][START_REF] Simmonds | The Digital Object Identifier (DOI)[END_REF])is now an ISO standard ("ISO 26324:2012 -Information and documentation -Digital object identifier system" 2012)and has been identified as one of the best choices among those complying with the definition of a digital identifier from a comparative evaluation based on seven criteria [START_REF] Khedmatgozar | A Basic Comparative Framework for Evaluation of Digital Identifier Systems[END_REF].

A DOI name consists of two parts [START_REF] Sidman | A practical guide to automating the digital supply chain with the Digital Object Identifier[END_REF][START_REF] Simmonds | The Digital Object Identifier (DOI)[END_REF]. The first part consistsofa prefix corresponding to a unique numeric string beginning with the numeral 10 assigned by the International DOI Foundation or by DOI registration agencies (e.g. CrossRef for scientific publishers)to the registrant that submitted the information about the digital object (publishers in the case of scientific publications). The second part consists ofa suffix corresponding to an alphanumeric string or series of strings assigned by the publisher and is used internally to identify the digital object.

The system operates in such a way that when a DOI is first assigned to an object, thepublisher registers its identity and current network location in the DOI system. Incoming requests for the DOI are resolved to the appropriate URL. When the object moves, the publisher of that contents simply updates its DOI record with the new URL.Hence, accessing digital objects using DOIs ensuresseamless and correct routing to the new and correct location of objects [START_REF] Sidman | A practical guide to automating the digital supply chain with the Digital Object Identifier[END_REF] andenables permanent and unambiguous identification and access to digital objects.One of the main benefits of referencing an object by a DOI versus a simple URL is persistence. When an object moves, its URL changes but its DOI remains the same. Furthermore, using DOIs instead of URLs allows multiple resolution. In addition to the DOI and URL, depositedmetadata may describe multiple formats of an article (pdf, html, XML) and several locations (e.g. publisher's website, PubMed Central). Therefore, end users can choose their preferred format or download location [START_REF] Derisi | The What and Whys of DOIs[END_REF]. The DOI can also be applied at any level of granularity (to a whole book or to chapters, illustrations or tables) or any file type (text, image, audio-video) [START_REF] Wang | Digital object identifiers and their use in libraries[END_REF].

With the establishment of DataCite in 2009, whose aims are to facilitate access to research data, to increase the acceptance of data publication, and to support data archiving [START_REF] Brase | The Tenth Anniversary of Assigning DOI Names to Scientific Data and a Five Year History of DataCite[END_REF][START_REF] Honor | Data Citation in Neuroimaging: Proposed Best Practices for Data Identification and Attribution[END_REF][START_REF] Neumann | DataCite and DOI names for research data[END_REF], DOIs can also be assigned to research data. During the five past years, DataCite has grown into a global consortium and has assigned over four million DOIs to scientific datasets [START_REF] Brase | The Tenth Anniversary of Assigning DOI Names to Scientific Data and a Five Year History of DataCite[END_REF].

DOIsofferother advantages for all those involved in the scientific community:

For publishers, paying one annual fee allowsaccess to articles via DOI links and functions even if content moves or changes ownership.DOIs also protect the copyright of published material [START_REF] Rosenblatt | The Digital Object Identifier: Solving the Dilemma of Copyright Protection Online[END_REF].The adoption of DOIs by publishers increases the discoverability of resources by search engines, increasing the traffic on websites: the number of site views for documents with a DOI were higher by an average of 66% using CrossRef compared to documents without the DOI service [START_REF] Sieck | Using the DOI to Improve Profitability In[END_REF]. Consequently, publishers increase their revenue. For example, for a medium-sized book publisher, the return on investment using the DOI could be as much as 12 times the cost of DOI implementation [START_REF] Sieck | Using the DOI to Improve Profitability In[END_REF].

For libraries, DOI links can extend accessto content not owned by the library and increase usage of acquired electronic resources for end-usersat no charge [START_REF] Wang | Digital object identifiers and their use in libraries[END_REF].

For researchers, DOIs provide access to over 19 million scientific objects available from over 20000 journals and from over 1500 publishers and companies ("crossref.org" 2016) bysimply using a copy/paste of the DOI in a DOI resolver (e.g.https://dx.doi.org/) or using bibliographic tools such as Zotero("Zotero | Home" 2016).Use of DOIs enriches end-user activity and the scholarly research process, and also guarantees persistent access and citability of articles.

For research funders, now that most of them require researchers to make their articles available in open access with different specifications (licenses and embargo periods), they need to track output from hundreds or even thousands of publishers. Thus, DOIscan offer a reliable way to help these institutions with their compliance processes("Crossref initiatives will support reporting to funders | Research Information" 2016).

DOIs can alsobe used in scientific production evaluation. For example, the National Council for Scientific and Technological Development (CNPq) in Brazil signed an agreement with ISI Thomson Reuters to allow access and online viewing of the number of citations in the Web of Science database for the articles registered in the Curriculum Lattes (a virtual platform integrating curricula databases, research groups, and institutions) with their respective DOIs [START_REF] Braile | After the Impact Factor, the DOI[END_REF].

Altmetrics are social web metrics for academic publications incorporating a number of variables such as view count, downloads, and comments in order to measure the impact of articles [START_REF] Galligan | Altmetrics: Rethinking the Way We Measure[END_REF][START_REF] González-Valiente | A review of altmetrics as an emerging discipline for research evaluation[END_REF][START_REF] Rasmussen | Altmetrics: an alternate perspective on research evaluation[END_REF][START_REF] Sud | Evaluating altmetrics[END_REF]. The DOI is an essential element for most sites or tools using altmetrics [START_REF] Galligan | Altmetrics: Rethinking the Way We Measure[END_REF]. For example, altmetric.com ("Altmetric" 2016), in order to disambiguate mentions of articles, searching for identifiers such as DOIsin web pages, PLOS Article-Level Metrics ("ALM" 2016)or PlumX ("Plumx" 2015)can create reports fromone DOI or from a list of DOIs. Furthermore, following the success of a DOI event tracker ("CrossRef's DOI Event Tracker Pilot -Crossref Blog" 2015; Tolwinska 2015), CrossRef will launch, in early 2017, the service CrossRef Event Data ("Crossref Event Data" 2016),which is an open data service based on DOIs. It will track activity surrounding a research work from potentially any web source where an event is associated with a DOI. This service will help researchers to follow the activity surrounding their articlesor research data. Publishers will able to use the service to track the dissemination of published articles to discover where they are being discussed, bookmarked, and linked. This will also help publishers to answer questions such as, "Is use growing over time?" or, "Which articles or subjects areas are being seen more than others?", considering multiple web sites rather than only their own web site [START_REF] Tolwinska | Need to Know What's Going On with an Article DOI? The Wait Is Over[END_REF].DOIs are also used as tools in some studies addressing altmetrics, e.g. ensuring the link between bibliometric databases and altmetric sites [START_REF] Haustein | Characterizing Social Media Metrics of Scholarly Papers: The Effect of Document Properties and Collaboration Patterns[END_REF]or identifying articles [START_REF] Haustein | Coverage and adoption of altmetrics sources in the bibliometric community[END_REF][START_REF] Thelwall | Do altmetrics work? Twitter and ten other social web services[END_REF].

As mentioned before, DOIs and the development of their related services are increasingly important for academia at different levels (articles, research data, etc.).But the same is true for bibliometrics and altmetrics, which justifies studies focusing on the assignment of DOIs to scientific articles.

To the best of our knowledge, only one study has evaluated the implementation of DOIsin the scientific literature, using Scopus -which indexes several databases, including Embase, Medline or Biobase [START_REF] Valderrama-Zurián | A systematic analysis of duplicate records in Scopus[END_REF] -and Web of Science Core Collection over the period 2005-2014[START_REF] Gorraiz | Availability of digital object identifiers (DOIs) in Web of Science and Scopus[END_REF]).This study showed that "there are still journals with a large number of items still lacking DOIs in 2014" and this "should be alarming for the corresponding editors and should give them reason to enhance the formal quality and visibility of their journals…". The authors also encouragescientists to review their publication strategies and to favour publication channels with established DOI assignments to result in a higher web presence and visibility".

The objectives of the present study are:

-to assess the DOI availability of articles in biomedical journals indexed in the PubMed database, year by year, over the period 1966-2015 (50-year retrospective study), -to evaluate the percentage of articles with DOIs in the PubMed database, published in 2015, -to complete these investigations with a geographical analysis of the countries and continents of publishers in order to identify the countries and continents which assign the most and the least DOIs to articles. To the best of our knowledge, this type of analysis has never been performed.

-to identify the most productive journals in the field of biology and medicine which did not yet assign DOIs in 2015.

Materials and methods

The search for papers to be included in this study was carried out from March 22nd to April 20th, 2016 using the PubMed database (http://www.ncbi.nlm.nih.gov/pubmed), developed by the National Center for Biotechnology Information (NCBI) at the National Library of Medicine (NLM). PubMed was chosen because it is the most widely used bibliographic database in medicine [START_REF] Falagas | Comparison of PubMed, Scopus, Web of Science, and Google Scholar: strengths and weaknesses[END_REF],and the country of publishers can be easily identified using the specific field "Country of Publication". However, contrary to other bibliographic databases (e.g. Scopus or Web of Science), any search tool is effective for distinguishing references with DOIs from others in PubMed. In PubMed, the interrogation of the field "Article Identifier" (which includes article identifiers submitted by journal publishers such as DOI) with the wildcard operator (e.g. 10* to extract all articles with a DOI) uses only the first 600 variations, because PubMed searches for the first 600 variations of a truncated term. It was then impossible to extract all references with DOIs using a query to assess the availability of DOIs in PubMed articles.Therefore, references were randomly selected from PubMed according to their PubMed identifier (PMID) using a Hypertext Preprocessor language (PHP) function that generates random integers (the PHP random number generation function called mt_rand (min,max), which returns an integer between min and max). The minimum value used was 1, and the maximum value 26995803, corresponding to the higher PMID assigned in PubMed at the time of the experiment was done. Then, the randomly generated integer was used to query PubMed by PMID using Efetch Entrez Programming Utilities, in order to extract the reference of the corresponding article. Data were downloaded from PubMed in Extensible Markup Language (XML) and were processed through developedPHPscripts. They werethen imported to Microsoft Excel 2013 (Microsoft, Redmond, USA) for data processing as done previously [START_REF] Boudry | Trends and topics in eye disease research in PubMed from 2010 to 2014[END_REF][START_REF] Boudry | Eye neoplasms research: a bibliometric analysis from 1966 to 2012[END_REF]. The analysis was limited to the publication type "Journal Article". For each article, the existence of a DOI was verified, then the date of publication, the name of the journal, and the country of publishers were analyzed. When the country of publisher was absent in the reference, it was determined using the NLM catalog ("Home -NLM Catalog -NCBI" 2016).

England, Scotland, Northern Ireland, and Wales were grouped into the United Kingdom.

Countries were clustered by their continent according to the United Nations classification("United Nations Statistics Division-Standard Country and Area Codes Classifications (M49)" 2016).

In order to assess implementation of DOIs year by year over the period 1966-2015, an analysis of 496665 randomly selected articles published from 1966 to 2015 was done, corresponding to 2.29% of the articles included in PubMed (the total number of articles in PubMed was 21680488on March 22nd 2016 for the same period). This first analysis was called "retrospective analysis" in this paper. the current implementation of DOIs(representing 21.68% of articles published in 2015 in the PubMed database). This second analysis was called "current analysis" in this paper. The year 2016 was not included because it was incomplete when the analysis was done, and a bias could have been introduced in the results when including journals with undefined frequencies of publication.

Results

Implementation of DOIs(retrospective analysis 1966-2015)

Among the 496665 articles studied, thenumber of articles with DOIs was 201055, corresponding to 40.78 % of articles with a DOI. In the period 1966-2015, we identified 115 countries of publishers. As shown in Table 1, among the 50 most productive countries of publishers, publishers from South Korea assigned the highest percentage of DOIs to articles, andpublishers from sevencountries assigned less than 1% of DOIs to articles they published over the period 1966-2015. The last group, with two countries, West Germany (-1990) and the USSR (-1991), had logically stopped their production in 1990 and 1991, when they ceased to exist, respectively. It is important to note that only publishers from three countries (the United States, Germany, and to a lesser extent the United Kingdom) made an effort to assign DOIs retrospectively before 1997, the yearDOIswerelaunched by the IDF. For continents,over the period 1966-2015the percentage of articles with DOIs varied from 15.23% (Latin America and the Caribbean) to 45.47% (North America) of articles with DOIs (Table 2). Results of countries and continents of publishers are presented in Table 3 and4

Discussion

To our knowledge, there are no similar studies examining the availability of DOIs in publications archived by PubMed, and such a study ofa geographical analysis of countries and continents of publishers has never beendone.PubMed was chosen because, contrary to other databases (Web of Science, Scopus), the country of publisher can be easily identified because of a specific field (called Country of Publication). Scopus did not provides this information, even when using the downloading format "all available information". The Web of Science provides a specific field called "Publisher Address" containing the complete address of the publisher, implying manual or automated isolation of the country from this complete address, which was not only impossible to carry out given the number of articles analyzed in the study, but could be a source or error.

Only one study, published in 2015 [START_REF] Gorraiz | Availability of digital object identifiers (DOIs) in Web of Science and Scopus[END_REF] The current analysis of articles published in 2015 showed that there were still 13.58% of articles that did not have DOIs, corresponding to 15.22% of journals indexed in PubMed which did not yet use DOIs. Publishers who do not assign DOIs must be made aware of the implications and realize, as some already have, the important role DOIs can play in identifying publishedarticlesmore easily as well as increasing their journal's impact [START_REF] Braile | After the Impact Factor, the DOI[END_REF]). More importantly, some scholars state that, in the near future, the value of a publication will be determined by the number of links to it [START_REF] Wang | Digital object identifiers and their use in libraries[END_REF]. Moreover, all of these articles and journals could be excluded from some scientific production evaluations or altmetric measurements, particularly that of the promising service CrossRef Event Data, whose objectives are to track Additionally, the list of the 20 most productive journals lacking DOIs for articles published in 2015 (current analysis), suggests that small and local publishers are mainly implicated. The cost of DOIs may explain the unwillingness of small publishers to implement them.

Nevertheless,publisher feesare proportional to the total publishing revenue, and the deposit fees per DOI (from 0.06 to 1.00 US dollarper content type) do not seem,in any case, to be an impenetrable barrier to the adoption of DOIs ("crossref.org : : publisher fees" 2016). Technical requirements to implement DOIs may also hinder small publishers who work in a traditional way.

Nevertheless, it is difficult to believe that in some countries, as in Russia, the 55 of 59 journals that have not implemented DOIs are small publishers, and that the cost and technical requirements could explain this finding. Only a qualitative survey among publishers in different countries would be able to clarify these results.The present study should also encourage publishers whohave not yet assigned DOIs to change their strategy in order to benefit from the numerous advantages offered by the system, as specified in the present article. They shouldunderstanding that with the new developments surrounding DOIs they may be increasingly on the fringes of scientific publication.

For publishers that have already adopted DOIs for their articles, a way to improve the efficiency of their utilization may be to assign several DOIs referring to different parts of articles (e.g. Researchers should be aware that choosing journals that assign DOIs is important,enabling their scientific communications to be more visible and accessible,and ensuring that they can benefit from new metrics such as altmetrics.Moreover, it increases the likelihood that their work will be read and cited.

The results presented in this article pertain only to biomedicine articles from PubMed and includes only articles (the study of [START_REF] Gorraiz | Availability of digital object identifiers (DOIs) in Web of Science and Scopus[END_REF] databases, which exist today [START_REF] Franceschini | Errors in DOI indexing by bibliometric databases[END_REF], should be limited.

  Fig. 1 shows the percentage of articles with DOIsfor articles published over the period 1966 to 2015. The percentage of articles with DOIs began to increasefor articles published around the year 2000, with spectacular growth in the years2002-2003,then reacheda peak in 2015.

Fig. 1 :

 1 Fig. 1: Percentage of articles with DOIsin the PubMed database for articles published over the period 1966-2015(retrospective analysis)

Fig. 2 :

 2 Fig. 2: Percentage of articles with DOIs in the PubMed database published over the period1966-2015: The ten most productive countries of publishers (retrospective analysis)

Fig. 3 :

 3 Fig. 3: Percentage of articles with DOIs in the PubMed database published from 1966 to 2015: continents of publishers

  ,has assessed the availability of DOIs in Web of Science Core Collection and Scopus over the period 2005-2014.This work showed that, in 2014, the percentage of citable documents (articles, reviews and proceeding papers) with DOIs in Scopus was about 86% in the health sciences, and 89% in life science. Our results are quite similar: 86.47% of articles had DOIs in PubMed in 2014. The retrospective analysis of availability of DOIs over the period 1996-2015 has shown that the percentage of articles with DOIs began to increase for articles published in and around the year 2000, with spectacular growth in the years 2002-2003,corresponding quite logically to the years following the launch of DOIs by the IDF.This showed that the DOI system was massively adopted by publishers in the years 2002-2003, meaning that most publishers rapidly understood the numerous advantages related to this system.It is noticeable that the highest percentages of articles with DOIs are mainly found in countries where large scientific publishers dominate (i.e.the United States, the United Kingdom, the Netherlands) and which have been identified by[START_REF] Vardakas | An analysis of factors contributing to PubMed's growth[END_REF]as the most productive countries of publishers in PubMed over the period 2004-2013. Of note is that unfortunately, overall,little effort has been made to assign DOIsretrospectively to articles published before 2000.This is regrettable because this retrospective assignment of DOIs could permit the enhancement of the value of former articles by facilitating their visibility and accessibility.

  activity surrounding articles or research data with DOIs from potentially any web source.The geographical analysis of countries and continents of publishers showed a high amount of heterogeneity in countries and continents. The analysis also demonstrated that seven of the 50 most productive countries of publishers assigned less than 10% of the DOIs to articles in 2015 (Israel, Malaysia, Mexico, Russia, Thailand, Ukraine and Serbia). For continents, in 2015 Asia was behind in both the percentage of articles with DOIs and the percentage of journals which had implemented DOIs. The reason for these observations is difficult to interpret because it is an arduous task to determinewhether recommendations for publishers to promote DOIs have been specifically implemented in countries or continents. It seems that few countries have implemented this type of recommendation[START_REF] Gorraiz | Availability of digital object identifiers (DOIs) in Web of Science and Scopus[END_REF].Globally, recommendations to promote DOIs can be found in "Recommended Practices for thePresentation and Identification of E-Journals (PIE-J)"("RP-16-2013 PIE-J (short URL) -National Information Standards Organization" 2013) published in 2013 by the National Information Standards Organization (NISO).Furthermore, the Policy Recommendations for the Open Access to Research Data in Europe (RECODE) project have also published such recommendations: "Publishers should require that data accompanying their publications are citable, and provide clear guidelines for data citation. Data citation should include DOIs" ("RECODE" 2015). As suggested by Park et al.[START_REF] Park | Examining success factors in the adoption of digital object identifier systems[END_REF], the further promotion of the core features of the DOI systems, perhaps through case examples and direction from industry/governmental leaders, are essential supporting factors in influencing the promotion of the DOI system's success. Industries and governments that develop DOI-compatible systems are encouraged to promote its features, which should be a powerful influence among organizations that are considering adopting DOI systems. This should have a significant influence on the countries identified in this study as assigning few DOIs, and may convince them to establish recommendations to encourage their publishers to consider using DOIs.

  tables, figures, videos, audio clips, datasets and supporting information), thereby allowing their access as separate entities(see as examples articles published by PLOS One)[START_REF] Derisi | The What and Whys of DOIs[END_REF].As in the case of CrossRef membership publishers("DOI display guidelines" 2016), suggesting researchers toroutinely mention DOIs in references in their instructions to authors would improve researchers' level of awareness of DOIs, and would also allow the readership to move from one article to another at the citation level, regardless of journal publisher. Without full-text citation linking, the user who discovers a desired resource while reading usually has to switch to a different search interface to locate and ultimately access that resource. With DOIs, it only takes a click or two to get to the full text, either as an authorized user or through pay-perview services("crossref.org" 2016).

  studied three types of publications: all document types from journals, proceedings series and conferences, and books extracted from Scopus and Web of Science Core Collection). These authors have shown that the social sciences and humanities have lower percentages of documents with DOIs compared to health,life, and physical sciences. It would be useful to complete their study by carrying out a geographical analysisas done in this study, particularly in the social sciences and humanities where practices and issues are different.Furthermore, as some studies have shown that publishing articles in open access journals increases their visibility and therefore increases the number of times they are cited in other articles[START_REF] Swan | The Open Access citation advantage: Studies and results to date[END_REF], future studies should explore whether there is a relationship between the assignment of DOIs and the number of citations received by articles.The advantages of developing standards for servicesare central, DOIs do not waver from this rule. Hence, the increasing use of DOIs should result in the multiplication of useful applications for all those involved in scientific research.In the future,DOIs should becomeincreasingly helpfultools. For this to continue, efforts must be made to generalize their use by publishers, without exception,and in allcountries and continents.This should have the consequence of familiarizing researchers with DOIs by means of articles they read, which will allow, in the near future, the development and extension of the use of DOIs to research data. However, errors in assigning DOIs,notably incorrect assignment of a single DOI to multiple articles by bibliometric

Table 1

 1 Number of articles published, number of articles with DOIs and percentage of articles with DOIs for the 50 most productive countries of publishers over the period 1966-2015 (retrospective analysis)

	Country of publisher	Number	Number	Percentage	Country of publisher	Number	Number	Percentage
		of	of	of articles	(continued)	of	of	of articles
		articles	articles	with DOIs		articles	articles	with DOIs
			with				with
			DOIs				DOIs
	United States	212668	98240	46.19	Norway		297	17.91
	United Kingdom	90291	51217	56.72	Korea (South)		1180	76.92
	Netherlands	27079	12770	47.16	Greece		303	20.21
	Germany (1990-)	19595	13117	66.94	Austria		414	29.34
	Japan	15713	1718	10.93	Hungary		176	15.00
	Switzerland	11588	4787	41.31	Romania		10	1.00
	France	11463	2068	18.04	Mexico		7	0.86
	West Germany (-1990)	10717	917	8.56	South Africa		44	5.53
	USSR (-1991)	9931	0	0.00	Yugoslavia (-2006)		0	0.00
	China	8254	1041	12.61	Egypt		438	65.96
					Czech Republic
	Italy	8083	807	9.98	(1993-)		16	2.43
	Denmark	5760	1438	24.97	Thailand		0	0.00
	Canada	5135	792	15.42	Israel		6	0.97
	Poland	4716	270	5.73	Iran		161	26.79
	India	4547	1768	38.88	Finland		9	1.52
	Ireland	4294	2354	54.82	Turkey		126	21.43
	Australia	4241	1408	33.20	Bulgaria		74	12.63
	Spain	3571	542	15.18	Singapore		112	23.43
	Russia (1991-)	3467	13	0.37	Pakistan		99	22.00
	Sweden	2965	389	13.12	Ukraine (1991-)		0	0.00
	Brazil	2712	602	22.20	Argentina		30	6.96
	East Germany (-1990)	2595	36	1.39	Chile		88	25.36
	Czechoslovakia (-1992)	2043	0	0.00	Saudi Arabia		47	15.31
					United Arab
	Belgium	1822	97	5.32	Emirates		41	13.62
	New Zealand	1760	710	40.34	Croatia (1991-)		20	7.25

Table 2

 2 Number of articles published, number of articles with DOIs, and percentage of articles with DOIs for continents of publishers over the period1966-2015 (retrospective analysis)

	Continent of publisher	Number of articles	Number of articles with DOIs	Percentage of articles with DOIs
	Europe	230740	92217	39.97
	North
	America	217803	99032	45.47
	Asia	34756	6355	18.28
	Oceania	6029	2118	35.13
	Latin
	America and
	the
	Caribbean	4917	749	15.23
	Africa	2420	584	24.13
	World	496665	201055	40.48

Table 3

 3 Number of articles published, number of articles with DOIs, percentage of articles with DOIs, number of journals, number of journals with DOIs, and percentage of journals with DOIsin the 50 most productive countries of publishersfor the year 2015 (current analysis)

						Number
						of
		Number	Number of	Percentage		journals	Percentage
		of	articles	of articles	Number of	with	of journals
	Country of publisher	articles	with DOIs	with DOIs	journals	DOIs	with DOIs
	United States	118291	105529	89.21	2810	2427	86.37
	United Kingdom	62944	61364	97.49	1724	1667	96.69
	Netherlands	14242	12893	90.53	479	439	91.65
	Germany	12014	11559	96.21	393	373	94.91
	Switzerland	10722	10266	95.75	248	236	95.16
	China	6169	1934	31.35	140	73	52.14
	India	4838	4124	85.24	168	145	86.31
	Japan	4497	3403	75.67	164	122	74.39
	New Zealand	3490	2495	71.49	124	119	95.97
	Italy	2840	1417	49.89	113	63	55.75
	France	2594	2186	84.27	96	84	87.50
	Canada	2065	1127	54.58	87	40	45.98
	Australia	2043	1509	73.86	83	66	79.52
	Korea (South)	1826	1733	94.91	120	119	99.17
	Ireland	1762	1710	97.05	38	36	94.74
	Brazil	1660	1646	99.16	60	59	98.33
	Russia	1582	33	2.09	57	2	3.51
	Poland	1412	875	61.97	72	47	65.28
	Spain	1329	1011	76.07	63	49	77.78
	Iran	1245	622	49.96	78	28	35.90
	Greece	1044	395	37.84	19	9	47.37
	Sweden	892	237	26.57	29	9	31.03
	Denmark	854	596	69.79	30	26	86.67
	Turkey	813	387	47.60	31	19	61.29
	Thailand	654	1	0.15	4	1	25.00
	Egypt	540	510	94.44	68	65	95.59
	Uganda	492	492	100.00	2	2	100.00
	Pakistan	466	74	15.88	10	4	40.00
	United Arab Emirates	445	117	26.29	38	5	13.16
	Czech Republic	364	48	13.19	20	5	25.00
	Romania	333	45	13.51	13	3	23.08
	Bulgaria	312	284	91.03	8	6	75.00
	Austria	302	299	99.01	19	17	89.47
	Hungary	274	220	80.29	16	9	56.25

Table 4

 4 Number of articles published, number of articles with DOIs, percentage of articles with DOIs, number of journals, number of journals with DOIs, and percentage of journals with DOIsby continent of publishers for the year 2015 (current analysis)

		Number	Number of	Percentage of	Number	Number of	Percentage
	Continent of	of	articles	articles with	of	journals with	of journals
	publisher	articles	with DOIs	DOIs	journals	DOIs	with DOIs
	North
	America	120356	106656	88.62	2897	2467	85.16
	Europe	117199	105856	90.32	3500	3100	88.57
	Asia	22061	12910	58.52	806	545	67.62
	Oceania	5533	4004	72.37	207	185	89.37
	Latin
	America and
	the
	Caribbean	2256	1783	79.03	94	73	77.66
	Africa	1385	1072	77.40	96	75	78.13
	World	268790	232281	86.42	7600	6445	84.80

Table 5

 5

lists the 20 most productive journals for articles published in 2015 (current analysis),based on the number of articles published,that did not assign DOIs to their articles.

Table 5

 5 The 20 most productive journals lacking DOIs: name of the journal, number of articles published, name of the publisher, country of publisher, ISSN and language of publication for articles published in 2015 (current analysis)

		Number				Language
		of		Country of		of
	Journal	articles	Publisher	publisher	ISSN	publication
	Int J Clin Exp Med	1187 e-Century Publishing	United States	1940-5901 English
	Int J Clin Exp Pathol	786	e-Century Publishing	United States	1936-2625 English
			American Scientific
	J Nanosci Nanotechnol	546	Publisher	United States	1533-4880 English
			Asian Pacific Organization
	Asian Pac. J. Cancer Prev.	504	for Cancer Prevention	Thailand	1513-7368 English
	Stud Health Technol
	Inform	466	IOS Press	Netherlands	0926-9630 English
			International Institute of
	Anticancer Res.	379	Anticancer Research	Greece	1791-7530 English
	Nippon Rinsho	282	Nippon Rinsho Co	Japan	0047-1852 Japanese
	Eur Rev Med Pharmacol
	Sci	257	Verduci Editore	Italy	2284-0729 English
	Zhonghua Yi Xue Za Zhi	224	Zhonghua yi xue hui	China	0376-2491 Chinese
	Zhongguo Zhong Yao Za
	Zhi	220	Zhongguo yao xue hui	China	1001-5302 Chinese
	Lakartidningen	204	Sveriges Lakarforbund	Sweden	1652-7518 Swedish
	Rev Med Suisse	204	Médecine et Hygiène	Switzerland	1660-9379 French
	Guang Pu Xue Yu Guang		Beijing da xue chu ban
	Pu Fen Xi	195	she	China	1000-0593 Chinese
			Den Alm Danske
	Ugeskr. Laeg.	180	Laegerforening	Denmark	1603-6824 Danish
			Lin chuang er bi yan hou
	Lin Chung Er Bi Yan Hou		tou jing wai ke za zhi bain
	Tou Jing Wai Ke Za Zhi	170	ji bu	China	1001-1781 Chinese
			Southeastern Surgical
	Am Surg	167	Congress	United States	1555-9823 English
	Huan Jing Ke Xue	166	Ke xue zhu ban she	China	0250-3301 Chinese
	Ned Tijdschr Geneeskd	162	Bohn Stafleu van Loghum Netherlands	1876-8784 Dutch
			Natural Product
	Nat Prod Commun	161	Communications	United States	1934-578X English
			Pakistan Medical
	J Pak Med Assoc	155	Association	Pakistan	0030-9982 English


Amélia Viricel

Benoit Simon-Bouhet

Laura Ceyrac

Violaine Dulau-Drouot

Per Berggren

Omar A Amir

Narriman S Jiddawi

Philippe Mongin

Jeremy J Kiszka
email: jeremykiszka@gmail.com

Habitat availability and geographic isolation as potential drivers of population structure in an oceanic dolphin in the Southwest Indian Ocean

Keywords: spinner dolphin, Stenella longirostris, genetic structure, microsatellites, mtDNA, East Africa

des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

Understanding factors influencing population connectivity has been a central and long-standing research avenue in marine ecology (e.g. [START_REF] Cowen | Connectivity of marine populations: Open or closed?[END_REF]2007;Selkoe et al 2008). Past studies have shown that the genetic structure of marine populations is driven by a number of processes, including water currents, sea floor topography, water temperature and life history (e.g., [START_REF] Fullard | Population structure of long-finned pilot whales in the North Atlantic: a correlation with sea surface temperature?[END_REF][START_REF] Fontaine | Rise of oceanographic barriers in continuous populations of a cetacean: the genetic structure of harbour porpoises in Old World waters[END_REF][START_REF] Pelc | Geographical patterns of genetic structure in marine species with contrasting life histories[END_REF][START_REF] Ciannelli | Small-scale genetic structure in a marine population in relation to water circulation and egg characteristics[END_REF][START_REF] Mendez | Molecular ecology meets remote sensing: environmental drivers to population structure of humpback dolphins in the Western Indian Ocean[END_REF][START_REF] Mendez | Integrating multiple lines of evidence to better understand the evolutionary divergence of humpback dolphins along their entire distribution range: a new dolphin species in Australian waters?[END_REF].

In cetaceans, factors that can lead to discontinuous relationships between genetic and geographic distance include habitat characteristics, intra-species niche partitioning (e.g. foraging specialization) and kinship, combined with demographic processes (e.g., [START_REF] Hoelzel | Evolution of population genetic structure in marine mammal species[END_REF][START_REF] Möller | Habitat type promotes rapid and extremely localised genetic differentiation in dolphins[END_REF][START_REF] Mendez | Molecular ecology meets remote sensing: environmental drivers to population structure of humpback dolphins in the Western Indian Ocean[END_REF], Louis et al. 2014a,b;Viricel and Rosel 2014).

Beside its fundamental importance in marine ecology, understanding the spatial structure and genetic connectivity of marine populations is also critical for conservation and management purposes. Indeed, while no marine ecosystem is completely unaffected by human activities, threats to populations of marine organisms vary geographically [START_REF] Halpern | A global map of human impact on marine ecosystems[END_REF]). In the Southwest (SW) Indian Ocean for instance, the importance of marine mammal bycatch is spatially variable and it seems to primarily affect inshore species, including coastal delphinids [START_REF] Kiszka | Marine mammal bycatch in the Southwest Indian Ocean: Review and need for a comprehensive status assessment[END_REF]. Thus, delimiting biologically meaningful conservation units (i.e., based on population structure assessments) will be a crucial step toward preserving the marine megafauna of this region.

The spinner dolphin (Stenella longirostris) is one of the most abundant and widely distributed tropical delphinids [START_REF] Perrin | Spinner dolphin Stenella longirostris[END_REF]. Four subspecies are currently recognised, based on morphological and ecological differences [START_REF] Perrin | Spinner dolphin Stenella longirostris (Gray, 1828)[END_REF][START_REF] Perrin | Spinner dolphins (Stenella longirostris) of the western Pacific and Southeast Asia: pelagic and shallow-water forms[END_REF]). The Gray"s spinner dolphin (S. longirostris longirostris), hereafter the spinner dolphin, is primarily an insular subspecies, and its distribution includes the Atlantic, Indian and Pacific Oceans [START_REF] Perrin | Spinner dolphin Stenella longirostris (Gray, 1828)[END_REF]. In French Polynesia, Hawaii and the Maldives, spinner dolphins enter atolls, sheltered bays and lagoons through reef channels in the morning and leave in the afternoon to feed offshore overnight [START_REF] Norris | The Hawaiian Spinner Dolphin[END_REF][START_REF] Anderson | Observations of cetaceans in the Maldives, 1990-2002[END_REF][START_REF] Gannier | Environmental variables affecting the residence of spinner dolphins (Stenella longirostris) in the Bay of Tahiti (French Polynesia)[END_REF], essentially on mesopelagic prey [START_REF] Perrin | Stomach content of porpoise, Stenella spp., and yellowfin tuna, Thunnus albacares, in mixed-species aggregations[END_REF][START_REF] Dolar | Comparative feeding ecology of spinner dolphins (Stenella longirostris) and Fraser"s dolphins (Lagenodelphis hosei) in the Sulu Sea[END_REF]. Around the lagoon of Mayotte, in the Mozambique Channel (SW Indian Ocean), spinner dolphins primarily inhabit the outer slope of the barrier reef to rest and socialise, and rarely enter the lagoon (Kiszka et al. 2010a[START_REF] Kiszka | Ecological niche segregation within a community of sympatric dolphins around a tropical island[END_REF]. During the past 20 years, extensive work has been conducted on the movements, behaviour, social, and genetic population structure of insular spinner dolphins, particularly in the Pacific Ocean [START_REF] Norris | The Hawaiian Spinner Dolphin[END_REF][START_REF] Karczmarski | Spinner dolphin in a remote Hawaiian atoll: social grouping and population structure[END_REF][START_REF] Oremus | Isolation and interchange among insular spinner dolphin communities in the South Pacific revealed by individual identification and genetic diversity[END_REF][START_REF] Andrews | Rolling stones and stable homes: social structure, habitat diversity and population genetics of the Hawaiian spinner dolphin (Stenella longirostris)[END_REF]. These studies highlight that spinner dolphins may form "fission-fusion" societies, with groups forming and separating over short periods of times, such as around the big island of Hawaii [START_REF] Karczmarski | Spinner dolphin in a remote Hawaiian atoll: social grouping and population structure[END_REF][START_REF] Andrews | Rolling stones and stable homes: social structure, habitat diversity and population genetics of the Hawaiian spinner dolphin (Stenella longirostris)[END_REF]. However, social structure may vary according to habitat characteristics and geographical isolation. Indeed, at the remote Midway atoll (Hawaii), spinner dolphins form stable groups with high level of site fidelity, limited emigration/immigration and strong inter-individual associations [START_REF] Karczmarski | Spinner dolphin in a remote Hawaiian atoll: social grouping and population structure[END_REF]. In this region, gene flow is more restricted among populations showing a fluid social structure (the Kona Coast of the island of Hawaii) than among populations with stable social groups (Midway and Kure Atolls) [START_REF] Andrews | Rolling stones and stable homes: social structure, habitat diversity and population genetics of the Hawaiian spinner dolphin (Stenella longirostris)[END_REF]).

In the southwestern Indian Ocean, the spinner dolphin is one of the most common small cetacean species in tropical and subtropical waters, particularly around islands and reef complexes off Zanzibar [START_REF] Amir | The incidental catch of dolphins in gillnet fisheries in Zanzibar, Tanzania[END_REF], Mayotte (Kiszka et al. 2010a[START_REF] Kiszka | Ecological niche segregation within a community of sympatric dolphins around a tropical island[END_REF] and the Comoros archipelago (Kiszka et al. 2010c), La Réunion (Dulau-Drouot et al. 2008[START_REF] Condet | Habitat selection of two island-associated dolphin species from the south-west Indian Ocean[END_REF], Madagascar [START_REF] Rosenbaum | Marine mammals of Madagascar[END_REF] and Mauritius (Webster et al. 2015). Spinner dolphins (particularly the S. l. longirostris form) are rarely observed in open ocean waters (> 2,000 m), but can occur for short periods of time between islands (particularly in island chains), mostly when undertaking overnight foraging trips (e.g. [START_REF] Kiszka | Ecological niche segregation within a community of sympatric dolphins around a tropical island[END_REF], Mannocci et al. 2013, Thorne et al. 2012). As this species occurs in coastal and reef-associated waters, spinner dolphins are impacted by human activities, including past hunting and bycatch off Zanzibar (Stensland et al. 1998[START_REF] Amir | Recent records of marine mammals in Tanzanian waters[END_REF], direct hunting and bycatch in south-western Madagascar [START_REF] Razafindrakoto | Preliminary assessment of cetacean incidental mortality in artisanal fisheries in Anakao, southwestern region of Madagascar[END_REF] and disturbance from dolphin-watching activities such as on the west coast of Mauritius (Webster et al. 2015). However, the geographical extent of the influence of such direct and indirect effects on populations is unknown.

This study aims to characterise genetic diversity and population structure of spinner dolphins in the SW Indian Ocean, particularly from Zanzibar (Tanzania), Mayotte (Comoros archipelago) and La Réunion (Mascarene archipelago) (Fig. 1). These islands were selected because they are located along a west-east gradient, from continental waters of Africa (Zanzibar) to the most isolated and remote oceanic islands (La Réunion). We also characterise depth preferences of spinner dolphins at these sites to evaluate their reliance on shallow-water habitat and to estimate habitat size around the two oceanic islands. Resting habitat availability has been suggested to influence population size and dispersal in other island-associated spinner dolphin populations [START_REF] Andrews | Rolling stones and stable homes: social structure, habitat diversity and population genetics of the Hawaiian spinner dolphin (Stenella longirostris)[END_REF]. We evaluate the relationship between our estimations of genetic diversity and habitat size: islands with more suitable habitats are expected to sustain greater population sizes, which would maintain greater genetic diversity. Geographic distances separating the coasts of the three sites are greater than 900 km. Considering previous knowledge gathered in the Pacific [START_REF] Andrews | Patterns of genetic diversity of the Hawaiian spinner dolphin (Stenella longirostris)[END_REF][START_REF] Oremus | Isolation and interchange among insular spinner dolphin communities in the South Pacific revealed by individual identification and genetic diversity[END_REF][START_REF] Andrews | Rolling stones and stable homes: social structure, habitat diversity and population genetics of the Hawaiian spinner dolphin (Stenella longirostris)[END_REF], we hypothesised that geographic isolation is a major driver of the genetic population structure of spinner dolphins in this region, and that sampled islands should contain genetically distinct populations.

MATERIAL AND METHODS

Sample collection and DNA extraction

In total, tissue samples collected from 69 individual spinner dolphins off Zanzibar (n=21), Mayotte (n=29) and La Réunion (n=19) were available for this study (Fig. 1). For Zanzibar, muscle tissues were collected from bycaught animals in drift gillnets between 2000 and 2004 and were stored frozen at -20°C. Samples from Mayotte and La Réunion were collected during dedicated biopsy surveys undertaken in territorial waters from 2006 to 2011 from small boats.

Biopsy attempts were made opportunistically, when groups and individuals were easily approachable and when conditions were optimal (Beaufort < 2, dolphins closely approaching the boat). Optimal weather conditions allowed stability of the research boat and better chances to sample the animals successfully and safely (Kiszka et al. 2010b). Blubber and skin biopsies were collected using a crossbow (BARNETT Veloci-Speed® Class, 68-kg draw weight and BARNETT Panzer V Class, 68-kg draw weight) with Finn Larsen (Ceta-Dart, Copenhagen, Denmark) bolts and tips (dart 20-mm long, 7-mm-diameter). Biopsy samples were preserved individually in 90% ethanol before shipping and subsequent analysis. Biopsy sampling was conducted under French scientific permit #78/DAF/2004 (September 10, 2004) and permit #032/DAF/SEF/2008 (May 16, 2008) for Mayotte and MC/2009/336 for La Réunion. Genomic DNA was extracted from ~ 25 mg of tissue (muscle or skin) using a Nucleospin Tissue kit (Macherey-Nagel) following the manufacturer"s protocol.

Microsatellite genotyping and mitochondrial DNA (mtDNA) sequencing Twelve microsatellite loci previously optimized for S. longirostris were genotyped (Table 1).

PCR reactions included 20 ng of genomic DNA, 0.5 U Taq polymerase, 0.25 mM dNTPs, 1.5 mM MgCl 2 , 1X PCR Buffer, 0.125 M of each primer in a 20 L final volume. PCR profiles were as follows: initial 5 min denaturation step at 94C followed by 35 cycles of 30 s at 94C, 30 s at a specific annealing temperature (see Table 1), 45 s at 72C, and by a final 7-min extension step at 72C. All PCRs were conducted in a Techne TC-5000 thermocycler. PCR products were visualized using polyacrylamide gels on the LICOR 4300 DNA Analyser. Allele sizes were determined by eye using a size standard, and by two different researchers to ensure consistency in scoring.

A portion of the mtDNA control region was amplified using primers Dlp-1.5 (5"-TCACCCAAAGCTGRARTTCTA-3") [START_REF] Baker | Mitochondrial DNA variation and maternal gene flow among humpback whales of the southern hemisphere[END_REF]) and Dlp-8G (5"-GGAGTACTATGTCCTGTAACCA-3") [START_REF] Dalebout | Worldwide structure of mtDNA diversity among Cuvier"s beaked whales (Ziphius cavirostris): implications for threatened populations[END_REF]. PCR reactions included 0.5 U Taq polymerase, 0.25 mM dNTPs, 1.5 mM MgCl 2 , 1X PCR Buffer, 0.125 M of each primer and 50 ng of genomic DNA in a 50 L reaction volume. PCRs were conducted in a Techne TC-5000 thermocycler using the following profile: initial 5 min denaturation step at 94C followed by 35 cycles of 30 s at 94C, 30 s at 54C, 45 s at 72C, and by a final 7-min extension step at 72C. PCR products were purified and sequenced by Genoscreen (Lille, France). Sequences were edited in Chromas sequence viewer v. 2.33 (Chromas Technelysium) and were aligned using BioEdit v.5.0.6 [START_REF] Hall | BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT[END_REF].

Microsatellite analyses

We tested for departures from Hardy-Weinberg or linkage equilibrium within each sampled site using Genepop v. 4.2 (Raymond and Rousset 1995) with 10,000 dememorizations, 1,000 batches and 10,000 iterations per batch. The sequential Bonferroni technique [START_REF] Holm | A simple sequentially rejective multiple test procedure[END_REF]) was applied to correct for multiple tests. The presence of null alleles and scoring errors was assessed using Micro-checker v. 2.2.3 (van Oosterhoot et al. 2004) within each site. We searched for potential duplicates within biopsied animals by comparing their multi-locus genotypes (i.e., searching for identical genotypes in the dataset, and for genotypes with less than three different alleles overall) using Genalex v. 6.41 [START_REF] Peakall | GENALEX 6 : genetic analysis in Excel. Population genetic software for teaching and research[END_REF]. The mitochondrial haplotypes of samples with matching microsatellite genotypes were compared to confirm they were duplicates of the same individual. We investigated whether related individuals were included in the dataset by calculating maximum-likelihood estimates of pairwise relatedness using ML-Relate [START_REF] Kalinowski | ML-Relate: a computer program for maximumlikelihood estimation of relatedness and relationship[END_REF]). To avoid biases in population inferences that could result from family structure [START_REF] Anderson | The influence of family groups on inferences made with the program structure[END_REF], we removed one individual from each pair of potential relatives, i.e., individuals showing a pairwise relatedness value greater than 0.45 (as in Viricel and Rosel 2014). Allele richness, observed and expected heterozygosity were calculated using FSTAT v. 2.9.3.2 [START_REF] Goudet | FSTAT (version 1.2.): a computer program to calculate F-statistics[END_REF]Arlequin v. 3.5.1.2 (Excoffier and[START_REF] Excoffier | Arlequin suite ver 3.5: a new series of programs to perform population genetics analyses under Linux and Windows[END_REF], respectively. These molecular diversity indices were calculated for the whole dataset and for each site separately.

Population structure was assessed using a Bayesian approach implemented with Structure v. 2.3.4 [START_REF] Pritchard | Inference of population structure using multilocus genotype data[END_REF], which infers the number of populations (K) present in a dataset based on assumptions of Hardy-Weinberg and linkage equilibria within populations. Analyses were conducted using the admixture and correlated allele frequencies models, with and without prior information on individual location (option "LOCPRIOR"). Including prior information on sample locations can improve population inferences, particularly when the level of population differentiation is weak or recent [START_REF] Hubisz | Inferring weak population structure with the assistance of sample group information[END_REF]). To verify that using prior information did not artificially result in distinct clusters, we conducted additional Structure runs with the LOCPRIOR option after randomizing the sample location information in the input file. Three randomized input files were created. All Structure runs included 300,000 Markov chain Monte Carlo iterations and a 50,000 step burn in. Ten replicate runs were performed for K values between 1 and 5. Convergence was assessed by examining alpha and likelihood plots, and by comparing individual membership probabilities across replicate runs. The best K was chosen by comparing mean log probabilities among K values, and when K = 1 was ruled out, by applying Evanno"s method using K [START_REF] Evanno | Detecting the number of clusters of individuals using the software STRUCTURE: a simulation study[END_REF].

To assess genotypic variation among individuals and among the three locations, we applied a principal component analysis (PCA) to the microsatellite data using the package adegenet [START_REF] Jombart | adegenet: a R package for the multivariate analysis of genetic markers[END_REF]) in R v. 3.1.2 (R Core Team, 2015). In the PCA, allele frequencies were scaled using the centring option. An analysis of molecular variance (AMOVA, [START_REF] Excoffier | Analysis of molecular variance inferred from metric distances among DNA haplotypes: Application to human mitochondrial DNA restriction data[END_REF] was conducted in Arlequin v. 3.5.1.2. [START_REF] Excoffier | Arlequin suite ver 3.5: a new series of programs to perform population genetics analyses under Linux and Windows[END_REF] to estimate genetic differentiation among the three islands. Pairwise F ST estimates were calculated and significance was assessed using 10,000 permutations. We tested for isolation by distance (IBD) by conducting a Mantel test comparing pairwise genetic distances (F ST /(1-F ST ), Rousset 1997) with logtransformed (base 10) geographic distances among sampling locations. Geographic distances between population pairs were calculated as the Euclidean distance between the approximate centres of the areas where samples were collected. The Mantel test was performed using IBDWS v. 3.23 [START_REF] Jensen | Isolation by distance, web service[END_REF] with 10,000 randomizations. Finally, we investigated the occurrence of private alleles in each population identified using Genalex v. 6.41.

MtDNA sequence analyses

Diversity indices (haplotype and nucleotide diversities) were calculated for each site using Arlequin. We used JModeltest v. 0.1.1 [START_REF] Guindon | A simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood[END_REF][START_REF] Posada | jModelTest : Phylogenetic model averaging[END_REF]) and the Akaike Criterion to determine the most appropriate model of substitution given our sequence alignment.

AMOVAs comparing the populations identified using Structure were performed in Arlequin.

Genetic differentiation was measured using both F ST and  ST . For  ST , distances between haplotypes were calculated using the model of substitution selected with JModeltest. Significance was assessed using 10,000 permutations. We evaluated IBD as described above for microsatellite data. A median-joining network was constructed using Network v.4.6.1.2 [START_REF] Bandelt | Median-joining networks for inferring intraspecific phylogenies[END_REF] with default parameters to represent relationships among haplotypes.

Habitat size

In order to relate patterns of genetic structure and availability of habitat used by spinner dolphins, we created spinner dolphin habitat maps based on their depth preferences around Zanzibar, Mayotte and La Réunion. . These data were used to determine habitat preferences of spinner dolphins using depth as the main variable. We chose to focus habitat analyses on depth as it was previously identified as one of the main explanatory variables explaining spinner dolphin distribution patterns in this region [START_REF] Kiszka | Ecological niche segregation within a community of sympatric dolphins around a tropical island[END_REF][START_REF] Condet | Habitat selection of two island-associated dolphin species from the south-west Indian Ocean[END_REF] and in other parts of the world (e.g. Thorne et al. 2012). Depth data were extracted from the GEBCO_2014 Gridded bathymetric dataset (30 arcsecond resolution) hosted on the British Oceanographic Data Center (http://www.bodc.ac.uk). We considered that the preferred habitat corresponded to the depth range where 95% of spinner dolphin observations were made. Thus, we excluded 2.5% of the deepest and 2.5% of the shallowest observations to determine i) the preferred depth range for Mayotte and La Réunion separately (we did not include Zanzibar in this comparison as observations from Zanzibar come from bycatch events and are thus not directly comparable to sighting data from the other two islands), ii) the overall preferred depth range for the three islands together. For the second calculation, group size information available for sightings around Mayotte was taken into account. Our reasoning was that using group size information (when available) better reflects the preferred habitat of these populations (i.e. observing large groups in an area carries more weight than observing a single individual). We computed the total area available within the depth range limits obtained for the three islands together, and mapped these areas for Mayotte and La Réunion. It was not computed for Zanzibar since available habitat within this depth-range is virtually infinite along the East African continental shelf relative to the two islands. The retrieval of individual depths, the computation of projected surfaces and the mapping were all performed with the marmap package v0.9.2 (Pante and Simon-Bouhet 2013) in R v3.1.2.

RESULTS

Microsatellite data

Each sample was genotyped at 8 to 12 loci. No significant departure from Hardy-Weinberg or linkage equilibrium was observed (after sequential Bonferroni correction) in the three sampled sites. Furthermore, no scoring errors were identified using Micro-checker. Possible null alleles were detected for loci EV94 and 415/416 in Mayotte; however, since this issue was restricted to one site, we kept these two loci in all analyses. A duplicate sample was identified (100% matching genotypes and haplotype) in La Réunion, where the same individual was biopsied twice. Five pairs of potential relatives were detected using ML-Relate: two pairs in La Réunion, one pair in Zanzibar, and two pairs in Mayotte. Only one individual from each pair was kept in population analyses conducted with both types of markers (mtDNA and microsatellites) to avoid the potential bias of including relatives. The final microsatellite dataset included 63 individuals (Table 2). The number of alleles per locus ranged from six to 25 (Table 1). Observed heterozygosity and mean allele richness were similar among the three sites (Table 2).

No population subdivision was found when Structure was run without prior information (Fig. 2a).

Conversely, genetic structure was detected when individual location was used as prior information. The most likely number of populations present in the dataset was three based on comparison of the mean log probabilities and K (Fig. 2b), and corresponded to the three sampled sites (Fig. 2c): within each site, all individuals were assigned to the same cluster with high ancestry proportions (q > 0.8, mean q = 0.95). Randomizing sample locations in the Structure input file and applying the LOCPRIOR option resulted in no structure being detected by the program (most likely K = 1; see Appendix 1).

The first principal component of the PCA separated individuals from Mayotte from the other two locations, while the third principal component showed two separate clusters for individuals from La Réunion and Zanzibar (Appendix 2). Some overlap in the PC space was observed among the three clusters. Together, the first three principal components explained 12.7% of the total genotypic variation. The AMOVA conducted among the three populations revealed relatively weak, but significant genetic differentiation (Overall F ST = 0.020 p < 0.0001; Table 3). The Mantel test conducted to examine IBD was not significant (r = 0.21, R 2 = 0.04, p = 0.51). Private alleles were detected within each of the three sites (Mayotte: 31; La Reunion: 9; Zanzibar: 11).

MtDNA data

The final control region sequence alignment was 720 bp-long and included 28 unique haplotypes (Genbank accession # XXXXXXXX-XX). The sequence alignment included 42 substitutions and no indels. The model selected using JModeltest was Tamura-Nei (Tamura and Nei 1993) with a gamma correction ( = 0.726). Analyses of mitochondrial sequences supported the population structure detected using microsatellite loci: significant differences in mitochondrial haplotype frequencies (after sequential Bonferroni correction) were observed among all sites using F ST (Overall F ST = 0.084 p < 0.0001; Table 4). However, none of the pairwise comparisons were significant when distances among haplotypes were incorporated in the AMOVA (i.e., using  ST , Table 4). The test for IBD was not significant (r = 0.96, R 2 = 0.93, p = 0.17). There was one shared haplotype between La Réunion and Zanzibar, and three shared haplotypes between Mayotte and Zanzibar. Mayotte and La Réunion had no haplotypes in common (Fig. 3). No obvious phylogeographic structure was observed on the haplotype network.

Habitat size

Comparing the depth distribution data of the two oceanic islands revealed that the preferred depth range of spinner dolphins around La Réunion (16 to 935 m) was larger than the one calculated for Mayotte (5 to 175 m). When all observation data are combined, the locations used by 95% of the individuals sighted around Zanzibar, Mayotte and La Réunion have a depth range of 9 to 162 m.

The total surface available around Mayotte and La Réunion within this depth-range (9 to 162 m) was 1,036 and 327 km², respectively (Figure 4). The habitat surface corresponding to spinner dolphins" depth preferences is thus three times larger in Mayotte than in La Réunion.

DISCUSSION

Marine organisms with high dispersal capacities can show weak genetic structure across large geographic distances. For instance, the short-beaked common dolphin (Delphinus delphis) and the Portuguese dogfish (Centroscymnus coelolepis) each form a single panmictic population across the eastern Atlantic (Veríssimo et al. 2011;[START_REF] Moura | Atypical panmixia in a European dolphin species (Delphinus delphis): implications for the evolution of diversity across oceanic boundaries[END_REF]. However, even low levels of genetic differentiation can correspond to restricted levels of dispersal in a demographic sense and can be associated with adaptive divergence (e.g., [START_REF] Knutsen | Are low but statistically significant levels of genetic differentiation in marine fishes "biologically meaningful"? A case study of coastal Atlantic cod[END_REF][START_REF] Aykanat | Low but significant genetic differentiation underlies biologically meaningful phenotypic divergence in a large Atlantic salmon population[END_REF], and therefore represent important findings in terms of conservation and management.

In highly dispersive marine organisms, incorporating spatial information as prior information in genetic analyses can help reveal genetic differentiation (Selkoe et al. 2008), as illustrated in the present study. Indeed, we did not detect any structure when using a Bayesian approach to detect the number of genetically distinct populations based on microsatellite data alone. Including prior information about sample location in the Bayesian analysis allowed retrieving three populations, corresponding to the distinct islands that were sampled. Our results are in accordance with expectations of [START_REF] Latch | Relative performance of Bayesian clustering software for inferring population substructure and individual assignment at low levels of population differentiation[END_REF] and [START_REF] Hubisz | Inferring weak population structure with the assistance of sample group information[END_REF], as the F ST estimates we obtained from analysis of microsatellite data fell right in the range where genetic structure can remain undetected without prior information (0.01 < F ST < 0.03). This approach (Structure with LOCPRIOR: [START_REF] Hubisz | Inferring weak population structure with the assistance of sample group information[END_REF] does not seem to falsely inflate genetic structure as shown in previous studies (e.g. [START_REF] Christie | Self-recruitment and sweepstakes reproduction amid extensive gene flow in a coral-reef fish[END_REF][START_REF] Russello | Detection of outlier loci and their utility for fisheries management[END_REF]Viricel 2012). Results from the three randomizations we conducted indicate the genetic clusters we observed using prior information are biologically significant (Appendix 1). Furthermore, the Mantel test comparing pairwise genetic and geographic distances was not significant, suggesting IBD did not confound our Structure results. The three distinct populations inferred from Structure and observed in the PCA based on nuclear data were supported by the significant genetic differentiation estimated with mitochondrial DNA sequences using F ST . Possible explanations for the lack of significant differentiation observed using  ST are that the observed genetic differentiation is recent and that not enough time has passed for new mutations to accumulate within populations; or that migration rates among these populations are greater than the mutation rate of the mitochondrial DNA control region. In these cases, incorporating distances among haplotypes in the AMOVA can increase noise in the analysis [START_REF] Bird | Detecting and measuring genetic differentiation[END_REF], rendering  ST less informative than F ST .

The preferred depth range of spinner dolphins we inferred from sighting and bycatch data (i.e., for the three locations analysed together) confirmed that the species is associated with relatively shallow-water habitat at these locations, which corresponds to their resting grounds. The wider depth range observed for the population of La Réunion (also described in [START_REF] Dulau-Drouot | Cetacean diversity off La Réunion Island (France)[END_REF]) may reflect the very narrow continental shelf of the island, with depth increasing rapidly from the shore, compared to Mayotte. In fact, a recent habitat modelling study showed that despite the wide depth range of spinner dolphin observations aroung La Réunion, most sightings occur between 51 and 63 m of depth, within a "core habitat" also characterised by flat and lightcolored seabeds [START_REF] Condet | Habitat selection of two island-associated dolphin species from the south-west Indian Ocean[END_REF]. Geographic isolation and the reliance of spinner dolphins on appropriate shallow-water resting habitat during daytime are likely factors causing and/or maintaining divergence between populations occupying Mayotte and La Réunion.

Although we cannot tease apart the relative role of each of these two factors, hypotheses can be made based on what has been observed in other small delphinid species. Indeed, large geographic distance from continental waters does not seem to be a sufficient driver to cause restricted gene flow in pelagic dolphins found around oceanic islands as illustrated by population structure studies on Atlantic spotted dolphin (Stenella frontalis) and common bottlenose dolphin (T. truncatus) populations from the Azores: the pelagic morphotypes within these two species form panmictic populations over large distances, from the Azores to offshore waters of the northwestern Atlantic at least 4500 km away [START_REF] Quérouil | High gene flow in oceanic bottlenose dolphins (Tursiops truncatus) of the North Atlantic[END_REF]Viricel & Rosel 2014). On the other hand, some pelagic island-associated delphinids show restricted gene flow across short distances within an archipelago such as common bottlenose dolphin populations around Hawaii [START_REF] Martien | Population structure of island-associated dolphins: evidence from mitochondrial and microsatellite markers for common bottlenose dolphins (Tursiops truncatus) around the main Hawaiian Islands[END_REF], or the rough-toothed dolphin (Steno bredanensis), which displays longterm site fidelity around the Society Islands (French Polynesia), and fine-scale genetic differentiation between two islands only 170 km apart [START_REF] Oremus | Pelagic or insular? Genetic differentiation of rough-toothed dolphins in the Society Islands, French Polynesia[END_REF]. Thus, daily reliance on near-shore, insular habitats may be a predominant driver of population structure in pelagic delphinids, even more so than geographic isolation.

The genetic subdivision we observed is consistent with other studies conducted on S. longirostris in the Pacific Ocean, which indicated that insular populations are generally discrete. Levels of genetic differentiation were similar to those observed among Society (French Polynesia) and Hawaii archipelago [START_REF] Oremus | Isolation and interchange among insular spinner dolphin communities in the South Pacific revealed by individual identification and genetic diversity[END_REF][START_REF] Andrews | Rolling stones and stable homes: social structure, habitat diversity and population genetics of the Hawaiian spinner dolphin (Stenella longirostris)[END_REF]. Spinner dolphins found around islands of the Society Archipelago (French Polynesia) form relatively closed communities showing strong island fidelity [START_REF] Oremus | Isolation and interchange among insular spinner dolphin communities in the South Pacific revealed by individual identification and genetic diversity[END_REF]). Gene flow among these communities is restricted, despite the short geographic distances separating some of these islands (i.e. tens of kilometers). [START_REF] Oremus | Isolation and interchange among insular spinner dolphin communities in the South Pacific revealed by individual identification and genetic diversity[END_REF] suggest that these communities are characterised by a metapopulation dynamics, which would explain the high genetic diversity and large island effective population sizes estimated from their molecular data. In the Hawaiian archipelago, finescale genetic structure is also observed, but patterns of gene flow vary according to social structure and habitat availability [START_REF] Andrews | Rolling stones and stable homes: social structure, habitat diversity and population genetics of the Hawaiian spinner dolphin (Stenella longirostris)[END_REF]. Overall, both the genetic and social structure of this species seems to be influenced by the availability and extent of resting areas [START_REF] Karczmarski | Spinner dolphin in a remote Hawaiian atoll: social grouping and population structure[END_REF][START_REF] Andrews | Rolling stones and stable homes: social structure, habitat diversity and population genetics of the Hawaiian spinner dolphin (Stenella longirostris)[END_REF]. In Hawaiian populations, genetic differentiation increased with geographic distance among islands. In the present study, the tests for IBD we conducted for both types of markers were not significant. We have to note, however, that these tests had low statistical power since only three populations were sampled. Thus, future studies analyzing samples from additional islands in the SW Indian Ocean would better allow testing for IBD. Additionally, photo-identification data would complement present findings, as site fidelity may constitute another factor driving population structure in spinner dolphins from the SW Indian Ocean.

The genetic diversity we measured (Table 2) was similar to what has been reported for this subspecies in French Polynesia [START_REF] Oremus | Isolation and interchange among insular spinner dolphin communities in the South Pacific revealed by individual identification and genetic diversity[END_REF], and was greater than the diversity observed in Hawaii [START_REF] Andrews | Rolling stones and stable homes: social structure, habitat diversity and population genetics of the Hawaiian spinner dolphin (Stenella longirostris)[END_REF]. Despite the genetic differentiation of spinner dolphins from La Réunion and Mayotte, the genetic diversity of these two populations is similar to the diversity of spinner dolphins from the coast of Zanzibar. Factors influencing their population genetic diversity include effective population size (linked to drift) and immigration rates. Given the differences in habitat size among the three sites we compared, the respective local population size of the populations occupying these sites may differ. Thus, we hypothesize that the similar levels of genetic diversity we observe in these populations likely reflect low but recurrent gene flow, which may be sufficient to maintain genetic diversity within island communities. Spinner dolphins from the SW Indian Ocean could thus be under a metapopulation dynamics, similarly to what findings from [START_REF] Oremus | Isolation and interchange among insular spinner dolphin communities in the South Pacific revealed by individual identification and genetic diversity[END_REF] indicate for populations in the Society Archipelago.

Alternatively, the genetic divergence we measured could be recent, and these populations may have so far retained ancestral polymorphisms.

The present study constitutes the first population structure assessment for the spinner dolphin in the SW Indian Ocean and our findings have important conservation implications. Indeed, the habitat preferences and patterns of restricted gene flow we identified suggest spinner dolphin populations found off Mayotte and La Réunion are demographically independent from each other. Their differentiation makes them potentially vulnerable if directly impacted by human activities, and spinner dolphins found off these two islands should therefore be treated as two distinct conservation units at the national level. Analysing samples from other islands (e.g. Madagascar, Mauritius) within this region would allow further evaluating the genetic isolation of these populations. Bycatch levels of spinner dolphins in gillnets off Zanzibar [START_REF] Amir | The incidental catch of dolphins in gillnet fisheries in Zanzibar, Tanzania[END_REF] should be considered as a cause of concern, and this issue highlights the need of a population structure assessment along the east coast of Africa. Protecting important resting habitat is an important step toward the conservation of insular spinner dolphin populations. Dedicated surveys help assessing whether current marine protected areas encompass such habitats (e.g. in la

  From 2004 to 2008, sighting data were collected around Mayotte (n = 168 sightings in 224 days of survey) and La Réunion (n = 51 sightings in 278 days of survey) during small-boat dedicated surveys (see Dulau-Drouot et al. 2008; Kiszka et al. 2011 for sampling protocols). For surveys undertaken around Mayotte and La Réunion, the sampling effort did not follow predefined transects and was not homogeneous. However, both surveys covered shallow inshore waters, the outer slope of the reef and deep oceanic habitats (depth > 500 m around Mayotte: Kiszka et al. 2011 and depth > 1,000 m around La Réunion: Dulau-Drouot et al. 2008). For Zanzibar, as sighting data were unavailable, we used geographic locations of bycatch events that were recorded during a bycatch monitoring program coordinated by the Institute for Marine Sciences, University of Dar es Salaam, based on Zanzibar between 2000 and 2007 (n = 27 records)

Figure 1 .

 1 Figure 1. Study area and sample locations. Sample sizes are indicated for each site. The 200 m

Figure 2 .

 2 Figure 2. Bayesian clustering analysis (Structure) results obtained from analysis of 12

Figure 3 .

 3 Figure 3. Median-joining network of 28 mitochondrial control region haplotypes observed in 63

Figure 4 .

 4 Figure 4. Maps showing the surface available for daytime resting for spinner dolphins around

Figures

  Figures

  Figure 2 742 K LnP(K) K -2885 na 2 1 -3579 3.4 3 -3078 2.2 4 -3007 5.7 5 -3086 na

Table 1 .

 1 Réunion: Dulau-Drouot et al. 2008) and habitat modelling studies (e.g. Thorne et al. 2012, Twelve microsatellite loci genotyped in this study. PCR annealing temperature (Ta), reference, number of alleles, observed (Ho) and expected (He) heterozygosity are given for

	Condet & Dulau-Drouot 2016) allow identifying key areas where new conservation efforts
	should be focused.

Table 2 .

 2 Mitochondrial DNA (mtDNA) and microsatellite diversity indices for Stenella longirostris from each site: N, sample size; No. haplo, number of haplotypes; π, nucleotide diversity; h, haplotype diversity; AR, allele richness; H o , observed heterozygosity; H e ,

	expected heterozygosity
			mtDNA			Microsatellites
		N	No. haplo π	h	N	Mean AR Ho	He
	Zanzibar	20 9	0.013 0.826	20 7.1	0.747 0.790
	Mayotte	19 14	0.015 0.965	27 8.2	0.773 0.835
	La Réunion 16 9	0.012 0.900	16 7.5	0.798 0.818

Table 3 .

 3 AMOVA results obtained from analysis of microsatellite data from 63 S. longirostris individuals. Pairwise F ST values are shown below diagonal and corresponding p-values above diagonal.

		Zanzibar	Mayotte	La Réunion
	Zanzibar	NA	<0.001*	<0.001*
	Mayotte	0.024	NA	0.009*
	La Réunion	0.025	0.013	NA
	*significant p-value after sequential Bonferroni correction

Table 4 .

 4 AMOVA results for mitochondrial DNA sequences. Pairwise F ST and  ST values are shown below and above diagonal, respectively.

		Zanzibar	Mayotte	La Réunion
	Zanzibar	NA	0.050	0.014
	Mayotte	0.058*	NA	0.066
	La Réunion	0.130*	0.067*	NA

*significant p-value after sequential Bonferroni correction

Acknowledgements

At Mayotte, samples and data from July 2004 to June 2006 were collected during a dolphin research project conducted by the Office National de la Chasse et de la Faune Sauvage (ONCFS, Game and Wildlife Service) and the Agriculture and Forestry Office (Direction de l'Agriculture et de la Forêt). From May 2007 to April 2009, data were collected during a joined programme of the University of La Rochelle, the Office National de la Chasse et de la Faune Sauvage and the Collectivité Départementale de Mayotte. DNA samples were prepared at the Molecular Core Facility at the University of La Rochelle. We would like to thank the Associate Editor and four anonymous reviewers for constructive comments on the manuscript.

Funding: Funding was provided by the Conseil Général de Mayotte, the Ministère de l'Energie, l'Ecologie, le Développement Durable et de la Mer (MEEDDM), the University of La Rochelle and the Office National de la Chasse et de la Faune Sauvage. The samples and data collected in Zanzibar was supported by funding grants from SIDA/Sarec. In Reunion, sampling was cofunded by DEAL-Reunion, BNOI and IRD, as part of the Southwestern Indian Ocean Fisheries Project (SWIOFP).

Compliance with ethical standards

Confict of Interest: The authors declare that they have no conflict of interest.

Ethical approval: All applicable international, national, and/or institutional guidelines for the care and use of animals were followed.