Agnès Thierry

Charlotte Cockram

Generation of high-resolution Hi-C contact maps in bacteria

Keywords: Chromosome conformation capture, Hi-C, 3C, Genome organization, Bacteria, Prokaryotes

During the past decade, Chromosome Conformation Capture (3C/Hi-C)-based methods have been used to probe the 3D structure and organization of bacterial genomes, revealing fundamental aspects of chromosome dynamics. However, the current protocols are expensive, inefficient, and limited in their resolution. Here we present a simple, cost-effective Hi-C approach that is readily applicable to a range of Gram-positive and Gram-negative bacteria.

Introduction

Hi-C relies on the proximity ligation of DNA fragments held together by cellular proteins and once processed, yields a snapshot of the 3D organization of the chromosome.

The protocol comprises of six basic steps: 1) Cells are crosslinked with a chemical fixative to covalently link chromosomal regions that are in close proximity; 2) Following lysis, chromosomal DNA is fragmented by a frequently cutting restriction enzyme; 3) DNA overhangs are then filled in, with the incorporation of a biotinylated nucleotide;

4) The DNA is then subject to blunt-end ligation under dilute conditions to favor intramolecular ligation; 5) Following reverse crosslinking, the DNA is purified and the biotinylated chimeric DNA molecules are enriched by streptavidin pull-down. 6) Finally, the Hi-C libraries are prepared for paired-end sequencing.

Despite several key improvements to eukaryotic Hi-C protocols during the past few years, including the development of commercial kits for non-specialists, the bacterial Hi-C protocol has remained largely unchanged. This means that prokaryotic Hi-C contact maps are still significantly limited in terms of the resolution they can offer. In this chapter, we have improved upon this by dissecting the different steps of published eukaryotic and prokaryotic Hi-C protocols, we identified several crucial areas where relatively simple modifications generated remarkably improved bacterial Hi-C contact maps and we used this information to generate an optimized Hi-C protocol (Figure 1).

The protocol is suitable for a range of Gram-positive and Gram-negative bacteria and is readily applied to other organisms such as archaea. The resulting prokaryotic Hi-C experiments are much more cost and time-effective compared to their predecessors but most importantly facilitate contact matrices that can be resolved at higher resolutions, revealing new features of the bacterial chromosome architecture (Figure 1).

Methods

Generation of a bacterial Hi-C library

This protocol facilitates the generation of a Hi-C library and the subsequent sequencing library within two days. However, there are several steps where the library can be stored at -20°C or incubations extended and these are indicated throughout the protocol. We recommend that users initially prepare no more than 4 libraries at a time, but the protocol can easily be scaled up once users become familiar with the process. Data analysis and methods for generating Hi-C contact maps have been explained previously [START_REF] Lioy | Multiscale Structuring of the E. coli Chromosome by Nucleoid-Associated and Condensin Proteins[END_REF]2].

Cell fixation

1. Grow bacterial culture in an appropriate medium until you have a total of ~2 x 10 8 cells growing in the exponential growth phase.

2. Chemically crosslink protein-DNA interactions by adding fresh formaldehyde (3% final concentration) for 30 min at room temperature with gentle agitation. 5. Carefully resuspend the cell pellet in 25 ml of 1x PBS and centrifuge again.

6. Resuspend the pellet in 1 ml of 1x PBS and transfer to a 1.5 ml microcentrifuge tube. Centrifuge again (4000 x g, 5 min, room temperature).

7. Carefully remove supernatant and store the cell pellet at -80°C. Pellets can be stored for approx. 12 months.

Hi-C library construction

1. Remove the cell pellet from the -80°C freezer and thaw on ice.

2. Resuspend the pellet in 1.2ml of 1x TE + cOmplete protease inhibitor cocktail and transfer to a 2 ml Precellys tube containing 0.5 mm glass beads. 12. Mix sample 10x by gentle pipetting. 11. Incubate for 5 min at room temperature and then place back onto the magnetic rack for 1 min.

Mechanically disrupt cells using the Precellys Evolution tissue homogenizer

12. Carefully remove the supernatant and transfer it to a new 1.5 ml microcentrifuge tube.

13. Check DNA on 1% agarose gel to determine the size of the final library and to check for the presence of primer-dimers (see Note 3).

14. Determine DNA concentration using the Qubit dsDNA HS kit on a Qubit Fluorometer.

15. Store the final sequencing library at -20°C. [START_REF]Gently mix the reaction and incubate with gentle agitation for 3h at room temperature. 3.1.3 Reverse crosslinking and DNA purification 1. Following ligation, add 20 µl 500 mM EDTA, 80 µl 10 % SDS, and 100 µl 20 mg/ml proteinase K to the Hi-C library. Add 20 µl 500 mM EDTA, 20 µl 10 % SDS, and 10 µl 20mg/ml proteinase K to the ND and D controls. 2. Incubate all samples at 65°C overnight to reverse formaldehyde-mediated protein-DNA crosslinks[END_REF]. Prepare libraries for paired-end sequencing using the 75 cycle High-Output Kit v2.5. according to Illumina's instructions.

Notes

The choice of the restriction enzyme and buffer is an important parameter of a

Hi-C experiment and can take some optimization. Some enzymes, such as DpnII (^GATC), are incompatible with many bacterial species because of Dam methylation. It is also important to determine the GC content and number of restriction sites within a particular genome, to check whether the required Hi-C resolution can be achieved. Currently, we recommend HpaII (C^CGG) with NEBuffer 1 (10X NEBuffer 1: (200mM Tris-HCl pH 7.5, 100mM MgCl2, 10mM DTT, 1mg/ml BSA) for most applications but if a genome is particularly AT-rich, or if a specific AT-rich region is under investigation, we recommend using MluCI (^AATT) with the same buffer.

2. For Precellys machines without the Cryolys cooling attachment, the program is: V6700: 9 x 20s, (30s pause) and the tubes need to be removed every 3 cycles and placed on ice for 5 min to stop the sample overheating and any subsequent degradation. Lysis can also be performed with lysozyme and will produce comparable results for Gram-negative bacteria. However, formaldehydecrosslinked Gram-positive bacteria, such as B. subtilis, can be difficult to lyse with lysozyme and in such cases, we recommend either disrupting cells with glass beads or a French Press [3,4] 3. See Figure 2 4. For efficient sonication, a maximum of 5 µg of DNA should be used (as determined by QuBit analysis), if the Hi-C library exceeds this, then an aliquot should be taken and the remaining DNA stored at -20°C, as a backup. Once the correct quantity of DNA is obtained, the sample volume is adjusted to 130 µl using 1x TE buffer.

5. The sonication parameters detailed in this protocol have been optimized for the amount of material used and the Covaris S220 ultrasonicator. Other sonicators will require additional optimization to ensure the average DNA fragment size is ~300bp.

6. Adapter ligation can also be performed using T4 DNA ligase (New England Biolabs) and performed either at room temperature for 2 hours or overnight at 16°C. (c) Hi-C contact matrix of WT V. cholera (5 kb bins). (d) Side-by-side comparison of the E.coli contact maps generated using either the new Hi-C or previously published 3C approaches [START_REF] Lioy | Multiscale Structuring of the E. coli Chromosome by Nucleoid-Associated and Condensin Proteins[END_REF] (1 kb bins). (e) Side-by-side comparison of chromosome 1 from V. cholera contact maps generated using either the new Hi-C or previously published 3C approaches [5] (1 kb bins). Fig. 2. Typical results obtained for the different steps of the bacterial Hi-C protocol. DNA extracted from fixed cells (ND) is digested with a 4-base cutting restriction enzyme for 3 hours at 37°C (D). The DNA ends are then filled in with a biotinylated nucleotide and blunt-end ligation performed for 3 hours at room temperature (L). Following reverse crosslinking, the DNA is purified, sonicated (S), and then prepared for sequencing using a modified 'on-bead' approach. The size and quality of the final library (FL) are then checked on gel before pairend sequencing.

4 . 4 .

 44 Variable temperature incubator (30 -37°C)5. Refrigerated centrifuge (for 50 ml falcon tubes) 6. Refrigerated centrifuge for 1.5 ml -5ml microcentrifuge tubes. 7. Precellys Evolution tissue homogenizer (Ozyme) 8. Precellys Cryolys cooling attachment (optional) Restriction enzyme and corresponding restriction enzyme buffer (see Note 1) 5. 30 U/µl T4 DNA ligase (Weiss units, Thermo Fisher Scientific) 6. 20 mg/ml Proteinase K in water (Eurobio) 7. 10 mg/ml DNase-free RNase A in water (Euromedex) 8. 37% Formaldehyde solution (w/v) (Sigma-Aldrich) 9. 2.5 M glycine (Filter sterilized) 10. 10% sodium dodecyl sulphate (w/v, SDS) solution (Thermo Fisher Scientific) 11. 10% Triton X-100 in water (Thermo Fisher Scientific) 12. 10x Ligation Buffer (-ATP): 500 mM Tris-HCl pH 7.5, 100 mM MgCl2, 100 mM DTT 13. 10 mg/ml bovine serum albumin (BSA, Sigma-Aldrich) 14. 100 mM ATP (pH 7.0, Sigma-Aldrich) 15. 500 mM EDTA (pH 8.0, Thermo Fisher Scientific) 16. 3 M sodium acetate (pH 5.2) 17. 10:9:1 phenol:chloroform:isoamylalcohol (pH 8.2, Interchim) 18. 100% ethanol (Thermo Fisher Scientific) 19. 70% ethanol (VWR international) 20. 1x TE buffer (pH 8.0) 21. cOmplete protease inhibitor cocktail (EDTA-free, Sigma-Aldrich) 22. 1x PBS 23. 2 ml Precellys tubes containing 0.5 mm glass beads (VK05, Ozyme) 24. Milli-Q water 25. Ultrapure Agarose (Thermo Fisher Scientific) 26. 10 mM dAGTP 27. Biotin-14-dCTP (Thermo Fisher Scientific) 28. DNA Polymerase I, Large (Klenow) Fragment (New England Biolabs) 2.3 Consumables for sequencing library preparation 1. 1.5 ml microcentrifuge tubes 2. Milli-Q water 3. Agencourt Ampure XP beads (Beckman) 4. 70% ethanol (VWR International) 5. 1x Tween Wash Buffer (TWB): 5 mM Tris-HCl pH7.5, 0.5 mM EDTA, 1M NaCl, 0.05% Tween 6. 2x Binding Buffer (BB): 10 mM Tris-HCl pH7.5, 1 mM EDTA, 2 M NaCl 7. 1x NEBuffer 2 (New England Biolabs) 8. Phusion 2x High Fidelity Master Mix (Thermo Fisher Scientific) 9. 1x T4 DNA ligase buffer (New England Biolabs) 10. T4 Polynucleotide Kinase (PNK, New England Biolabs) 11. T4 DNA polymerase (New England Biolabs) 12. Klenow Fragment (3'-5' exo-, New England Biolabs) 13. DNA Polymerase I, Large (Klenow) Fragment (New England Biolabs) 14. 10mM dNTPs 15. 10mM dATP 16. Quick Ligation Kit (New England Biolabs) 17. Ultrapure Agarose (Thermo Fisher Scientific) 18. Dynabeads MyOne Streptavidin C1 (Thermo Fisher Scientific) 19. Qubit dsDNA HS assay kit (Thermo Fisher Scientific) 20. microTUBE AFA Fiber Pre-Slit Snap-Cap (Covaris)

3 .

 3 Quench formaldehyde by adding glycine (0.5 M final concentration), Incubate for 20 min at room temperature with gentle agitation.

4 .

 4 Transfer culture to a 50 ml falcon tube and collect cells by centrifugation (4000.x g, 10 min, room temperature).

(

  V7500: 5 x 30s, 20s pause (see Note 2).4. Carefully transfer the lyase (~1 ml in volume) to a 5 ml microcentrifuge tube, avoid transferring any of the glass beads.

10 .

 10 Airdry pellet for 1-5 min to remove residual EtOH but don't allow beads to overdry.11. Remove beads from the magnet and resuspend in 50 µl of 10 mM Tris-HCl pH 8.

7 .

 7 Adapters can be interchanged with any Illumina-compatible or custom sequences. We have previously used TruSeq DNA CD Indexes (Illumina) and Nextflex 48 barcodes (Perkin Elmer).

Fig. 1 (

 1 Fig. 1 (a) Overview of the Hi-C protocol. Normalized Hi-C contact maps of asynchronously

Figure 2

 2 Figure 2

Acknowledgments

We thank Romain Koszul and the members of the RSG lab for insightful discussions regarding the development of this protocol. This research was supported by funding to Romain Koszul from the Agence Nationale pour la Recherche (HiResBac ANR-15-CE11-0023-03) and from the European Research Council under the Horizon 2020 program (ERC grant agreement: 260822).


Tatiana Nikitina
email: tatiana.nikitina@cnrs.fr

Missionary descriptions of Mande languages: Verbal morphology in 19th century grammars

INTRODUCTION

Up to the mid-20th century, a large proportion of descriptive work on African languages was done by missionaries, who are still responsible for a significant proportion of linguistic research in some parts of Africa. Yet the role missionary linguistics has played in the development of modern African studies is rarely discussed. Just as the intricate relationship between academic and missionary linguistics remains controversial (see [START_REF] Dobrin | Practical language development: Whose mission?[END_REF] and other papers in the same volume), a controversy surrounds methods used by missionary linguists and their attitudes to the languages they encountered. On the one hand, while describing African languages, missionaries relied to a great extent on the models that were familiar to them. Interpreting their material in terms of categories of European languages, they sometimes oversimplified and distorted the data, and it is now common to assume that early missionary grammarians suffered from a lack of broad typological perspective and relied slavishly on the Graeco-Roman descriptive tradition.

On the other hand, it would be a mistake to regard the Graeco-Latin model as a fixed framework defying any modification. Challenged with data from languages strikingly different from Greek and Latin, missionary grammarians quickly became aware of the familiar model's inadequacy. They were faced with the difficult choice of either distorting their data or extending the model, and they did their best to find a compromise solution. In this respect, the attitude of some missionary linguists was perhaps even "more open-minded than that of contemporary grammarians in Europe" (Zwartjes 2011: 10): they undertook the task of "re-semantisation of traditional terminology as words were applied for other purposes" (Zwartjes 2011: 16).

In spite of the prominent role of missionary linguists in shaping the field of modern African linguistics, the approaches adopted in their early grammar descriptions remain virtually unstudied, just as the descriptions themselves are largely ignored by modern linguists. This study explores the ways two 19th century missionary grammarians approached the task of describing verbal morphology of two languages from the Mande family. Mande language are spoken in West Africa, and are commonly albeit not universallybelieved to be part of the Niger-Congo family [START_REF] Kastenholz | Comparative Mande studies: State of the art[END_REF]. The sources from which I draw examples are John Kemp's grammar of Susu, published in 1802 in Edinburgh ("A Grammar and Vocabulary of the Susoo Language"), and R. Maxwell MacBrair's grammar of Mandingo, published in 1842 in London ("A Grammar of the Mandingo Language"). The two authors worked independently and adopted different approaches to language description. While Kemp's work was commissioned by the Society for Missions to Africa and the East and was based on 2,5 years of fieldwork in what now is Guinea, MacBrair's grammar, printed for the Wesleyan-Methodist Missionary, was mostly written in England based on work with a language consultant. Neither description has, to my knowledge, been previously studied from the methodological point of view; their comparison, moreover, reveals interesting trends that seem to characterize missionary approaches to describing West African languages more generally.

Based on the examples of these early grammars, I illustrate the diversity of attitudes and approaches to language description concealed behind the label "missionary linguistics". Even in the narrow domain of verbal morphologywhich is far from prominent in the isolating Mande languagesthe two grammarians resort to very different strategies for coping with the otherness of the material they describe. Their approach also differs in certain ways from the approach that has become prevalent in modern descriptive studies. I address possible reasons behind that difference, and suggest that the contrasting approaches are explained in large part by different interests and goals of missionary and academic linguists.

Early missionary grammars are often looked down upon as inadequate from the point of view of both theory and method. However, 19th century missionaries can hardly be considered untrained or naive in matters of language description. Many early descriptions of African languages were written by experienced and thoughtful grammarians who were closely familiar with the practical difficulties of fieldwork and fully aware of its methodological challenges, The excerpt in (1), from Kemp's grammar of Susu, attests to the author's reflection on methodological difficulties, some of which were only solved with the invention of recording equipment:

(1) "With the greatest humility, and most earnest entreaties, he [a missionary] must beseech the rudest and most uncivil barbarians, to repeat a word ten, or perhaps twenty times, that he may catch its proper sound, and commit it to writing. When he inquires into its meaning, perhaps he may be shocked with its obscenity, or he may discover that the person who expressed it, was cursing him or threatening his life." (Kemp 1802: xiiixiv) In (2), the same author demonstrates acute awareness of the psychological challenges of fieldwork (which are rarely addressed even in modern fieldwork manuals, for no solution has so far been discovered):

(2) "Perhaps he may scarcely have attempted to ask a drink of water, when all who hear him will burst into a roar of laughter at his awkwardness. <…> Instead of being treated with respect, on account of his patience and constant labour, a missionary must lay his account, for being looked on as weak, silly, and deranged, by those, whose senselessness and stupidity will cause more than half his troubles." (Kemp 1802: xiv) The tendency to dismiss early missionary grammars as inadequate from the modern point of view can also hardly be explained by the missionaries' over-reliance on the European tradition of grammar description. Both authors show awareness of the fact that the Graeco-Latin framework is not fully applicable to the languages they are working with. In (3a), MacBrair makes it clear that he is consciously forcing his data into a framework to which it is inherently alien. In (3b), Kemp suggests a way of accommodating the classical paradigm to a conjugation-less language: instead of listing full paradigms, he only lists one form for each tense.

(3a) "By throwing the various verbal forms into some sort of a classical arrangement, they may be stated as follows…" (MacBrair 1842: 14) (3b) "In giving examples further, I conceive it to be unnecessary to set down any more than the first person of each tense, as the terminations of verbs are never altered…" (Kemp 1802: 25) All this shows that early grammarians were far from naive in fieldwork methods, and they were fully capable of noticing meaningful differences between their language of study and their reference languages (Latin, Greek or English). Why then are their grammars rarely perceived, by academic linguists, as part of the modern descriptive tradition? The real reason seems to lie in the specific missionary perspective on the ultimate goals of describing African languages, which differed strikingly from the perspective taken by modern academic linguists. While present-day fieldworkers value and cherish linguistic diversity, missionary grammarians describing Mande languages commonly viewed it as an annoying obstacle to the propagation of Christian values. In (4), for example, Kemp goes as far as to deplore the practical impossibility of eradicating local languages:

(4) "Some seem to be of opinion, that barbarous languages ought to be rooted out, and better ones introduced in their stead; but this is not so easily done as many suppose. The British have never yet been able to root out the language of the Highlands of Scotland, although efforts have been made for this purpose, particularly by the Society in Scotland for Propagating Christian Knowledge. <…> Many religious, political, and commercial considerations, might urge the British to endeavor to root out the language of the Highland people, and they may attempt it <…> yet, it may still require centuries to accomplish this desirable object." (Kemp 1802: xii-xiii) In the following sections I review in more detail some consequences of this attitude for the ways the two missionaries describe the verbal systems of Mandinka and Susu. I point out some shared features of the two descriptions (Section 2) and argue that they stand in sharp contrast to their very different approach to the otherness of African languages. I then discuss ways in which the two descriptions are different (Section 3). Section 4 concludes the paper.

SHARED FEATURES OF MISSIONARY GRAMMARS

Descriptions as learner grammars

Missionary grammars and dictionaries were written for beginner learners who arrived from Europe and were trained in the Graeco-Latin tradition; "[m]ethodologically speaking, these grammarianslexicographers worked as translators" (Zwartjes 2011: 11). This practical orientation is often reflected in explicit reference to language learners, as in (5a), and sometimes in encouraging statements such as (5b): (5a) "I shall endeavour to form as many tenses as I conceive to be of any use, to those who may wish to learn the Susoo language." (Kemp 1802: 19) (5b) "Nothing but close attention to the conversation of the Susoo people, can give any one a correct idea of their various arrangements of words. .<…> yet he has no cause to be discouraged from learning Susoo on this account, for it is certainly an easy language…" (Kemp 1802: 32) To make it easier for a beginner learner to grasp the basics of the language, the grammarians tried to present their data in a clear and accessible fashion, sometimes at the expense of data quality. The search for a compromise sometimes led to solutions that look unacceptable or excessive from the point of view of modern language description standards. The passage in (6), for example, suggests that the author deliberately manipulated both the grammar and the lexicon of the language, to make it look more familiar to the European learner:

(6) "Although some of the grammatical rules that I have laid down, may make the language less familiar to the Susoos, than when it is spoken with all the license which they use in common conversation, yet I have been so careful to make what I have written plain and easy, that I have not inserted a single phrase, which did not seem to be perfectly understood by a Susoo boy who dwells along with me, after I had told him the meaning of the Arabic words that I have introduced." (Kemp 1802: xli)

While lexical modification was apparently limited to the insertion of Arabic words, it is not entirely clear which aspects of Susu grammar were deemed inappropriate and were edited out of the description. What is clear, however, is that the author made a conscious decision to construct an artificial language that would both be understood by the Susus and correspond to his own idea of a language worth describing and learning.

The practical orientation of the descriptions had another methodological consequence. Since missionary grammars of West African languages were intended to be used by Europeans, primarily for the purposes of Bible translation and preaching, they tend to focus on difficulties of translating from English or French into a local language. Differences between the two languagesespecially lexical onesare commented upon from the point of view of a European trying to translate into the local language (7a-c); difficulties that may arise while translating from a local language are normally not mentioned.

(7a) "The deficiency in number and variety of the Prepositions causes no little difficulty in translating European phrases into Mandingo." (MacBrair 1842: 29) (7b) "It is impossible for me to say, whether they use a passive voice through all the modes and tenses that may be formed; but I am certain that an European may do so, by the help of an auxiliary verb, and be perfectly understood by the Susoos." (Kemp 1802: 19) (7c) "The Mandingoes have few proper adverbs; and although many words are necessarily used in an adverbial sense, yet they can scarcely be said to exist as a distinct and separate form of speech. It must be explained, however, in what manner the adverbs of foreign languages are rendered in Mandingo." (MacBrair 1842: 27)

Descriptions as a tool for language improvement

While instruction of newly arrived Europeans was a common way early grammars were put to use, the ultimate goal of linguistic efforts was Bible translation. Preaching and translating into a local language was an essential part of disseminating Christian values and improving the people's lives. It was widely accepted among missionaries to West Africa, however, that local languages in their current state of development were not yet suited for Bible translation. The task of a missionary linguist was to work on the improvement of the language, which was intimately related to the improvement in the people (8a,b).

(8a) "It is utterly impossible to translate the whole of the Scriptures, in a satisfactory manner, (to my mind,) into any of the native languages of Western Africa, till a greater degree of knowledge be spread amongst the inhabitants. These languages are at present inadequate to convey a proper sense of scriptural truth; and language can only improve with a proportional improvement in the people." (MacBrair 1842: 49) (8b) "The author expects that improvements in the language will take place, in conjunction with the gradual advancement of the people in knowledge and refinement." (MacBrair 1842: vii)

What was the missionaries' idea of a "good" language? What did they seek to improve in an African language? First and foremost, missionary grammarians working with Mande languages aimed at eradicating all kinds of irregularity. Strict exceptionless rules were valued and enforced wherever possible. The passage in (9) reflects the missionary belief in the prescriptive power of their work, in the effect it has on the way the language is actually spoken: failure to enhance regularity is described as potentially harmful to the language, as it could make matters worse instead of improving them. ( 9) "Before new words can be properly introduced into a language, it ought to be reduced to grammatical principles. Were this not attended to, there would be no end of its irregularities, and missionaries, instead of improving a language, would make it a great deal worse. Certain rules must be laid down, according to which, it must be both spoken and written." (Kemp 1802: xii) Consistent with the active promotion of regularity, exceptions to rules are hardly ever mentioned, and never listed. This might explain why so little attention is paid to lexical matters: the language improvement project was to eventually eliminate all irregularity, including lexically conditioned rules. The example in (10) illustrates the authors' attitude to exceptions, which are never explained or treated in detail.

(10) "Custom alone can teach the use of these verbs." (MacBrair 1842: 24) While the lack of interest in exceptions is characteristic of early descriptions, later ones, including those by missionaries, sometimes approach irregularity as a natural part of grammar. The examples in (11), from Duport's grammar of Susu, emphasize the variety of options the natural language offers: (11) "In some cases the verb with la affixed signifies the agent <…> The prefix wa sometimes turns a verb into a noun <…> Sometimes it appears to have the force of the Latin 'per' in continuing or strengthening the action <…> The prefix ra often converts a noun or adjective into a verb" (Duport 1865: 8) Striving to enforce regularity, missionary linguists did not limit themselves to exceptionsthey also sought to do away with dialectal diversity, which was considered annoying. Dialectal variation was viewed as an obstacle to language improvement, and standardization was perceived as a necessary step on the way to promoting regularity: (12a) "Nor must it be forgotten, that as there can be no regular standard in an unwritten language, so every native esteems his own way of speaking to be the best. In this respect also, the pretensions of individuals have been set aside, and those forms of expression are adopted which are most agreeable to the laws of construction; that so, these first attempts at Mandingo composition may be the commencement of a series of improvements in a language which is by no means destitute of harmony and elegance." (MacBrair 1842: vi) (12b) "What kind of English grammar should we have, were all the provincial irregularities introduced into it? It would be no more whimsical to write a general grammar, comprehending all the languages of the world, with all their irregularities, than to attempt to write a grammar of this kind. My object has been to lay down principles for making the language easy and regular, which are familiar to the Susoo people." (Kemp 1802: 53) While reducing the number of local African languages was obviously not in the power of a missionary linguist (cf. 4), a missionary could hope to reduce variation within a language through standardization. The most efficient tool in that enterprise was literacy. The role assigned to standardization in eliminating irregularity may explain its importance for the language improvement project.

(13) "[W]ere books written in heathen languages, the style in which they were written would become the standard, and no regard would be paid to every particular dialect, any more than in England." (Kemp 1802: xii) Another aspect of language valued by missionaries almost as highly as regularity was often referred to as "clearness" or "precision". In MacBrair's description, both terms appear along with regularity of form as objects worth studying in a language: (14) "The inhabitants of the interior use a more elliptical method of speaking than those on the coast; but the author would not have been justified in making choice of such a dialect as would tend to degrade the language. It has been his aim to study clearness, precision, and a regular grammatical form; so that whilst these works may be understood by all the Mandingoes, they may also be the means of giving them a habit of precision in the manner of their daily intercourse." (MacBrair 1842: i)

Both clearness and precision, however, are relative notions. They are defined with respect to the reader's expectations, and those expectations are formed by previous linguistic experience. One aspect of clearness that was of particular importance to MacBrair involved the use of overt expression in cases where overt markers appear in European languages. In (15), for example, the dialects described earlier as degrading the language (see 14) are said to omit pronouns and the possessive marker, and to make extensive use of "abbreviated" forms:

(15) "The greatest difference of dialect existing among the aborigines of the Gambia, proceeds from the frequent omission of the possessive and personal pronouns, the neglect of the sign of the possessive case, and the abbreviation of familiar words and phrases." (MacBrair 1842: vi)

In ( 16), MacBrair laments the tendency to omit conjunctions, pointing out that foreign intervention is necessary to eliminate this defect. ( 16) "The reason of this want of conjunctions proceeds from the circumstance of the natives always employing short sentences and phrases, the connection of which takes place only in the mind, and not by conjunctions copulative. <…> This is a great defect in the language, which only time and converse with foreigners can remedy. At present, however, such conjunctions must be omitted in translations, since the use of them is perplexing to the natives. The conjunction barri, "but", may be pretty largely employed after the manner of the Greek de." (MacBrair 1842: 33)

Finally, missionary grammars often share one more feature that likely derives from their practical orientation: unlike many academic grammarians of the time, missionaries pay relatively little attention to the significance of the particular language in a local economy or its prominence in local social structures. From the point of view of propagation of Christian values, the economic position and political relevance of a language is of little significance, cf. the relatively succinct description by MacBrair in (17a). Authors of academic, military and diplomatic occupation, on the other hand, often go to great lengths describing the exceptional qualities and military achievements of the people who speak the language, presumably justifying the efforts spent on the study of that language, cf. the descriptions in (17b,c).

(17a) "The Mandingo is one of the most extensive languages of Western Africa. It not only prevails in various parts of the coast, south of the Senegal, but reaches interiorly towards the Niger and the mysterious Timbuctoo. It is spoken by all the native tribes settled on the banks of the Gambia, and has been adopted by the roving hordes also which frequent the neighbourhood of this noble river." (MacBrair 1842: v) (17b) "It is one of the most important and extensively-used Languages in West Africa. It is the Language of a conquering Race, and has an expansion beyond its natural boundaries, though in its time it has suffered from the encroachment and domination of the Fulah of the Nuba-Fulah Group. The Mande-nga occupy a mountaineous Region, but spread far beyond their natural boundaries, conquering and assimilating weaker and inferior Races. <…> The Mande-nga are a fine and superior Race, and for the most part Mahometan, though some of the subjected tribes are still Pagan." (Cust 1883 on Mandinka) (17c) "Mais, d'une intelligence bien supérieure en général à celle des peuplades autochtones, d'un esprit plus ouvert et plus cultivé aussi par suite de leur conversion à l'islam, les Dyoula ont acquis, par des moyens d'ailleurs tout pacifiques, la prépondérance politique; leur langue s'est répandue parmi les indigènes de toute la région et est devenue pour ainsi dire la langue officielle des chefs et des notables, en même temps qu'elle devenait la langue diplomatique et commerciale dont usent entre elles les diverses tribus sur lequelles les Dyoula exercent une sorte de protectorat moral." (Delafosse 1901: 4) To sum up, missionary grammars show a number of shared features that can be explained by their common practical orientation. As the grammars were written for educated Europeans intending to translate into a local language, they show strong translation bias and often refer explicitly to the reader. As the grammars were also an important tool in language policy, they promote emphatically the idea of language improvement by encouraging regularity, clearness, and precision. Unlike nonmissionary descriptions, they show little interest in the people's moral qualities or military achievements.

DEALING WITH OTHERNESS: DIVERSITY OF MISSIONARY APPROACHES

Three types of challenge

The previous section addressed some properties that characterize missionary grammars of Mande languages and which were shown to derive from a very specific common perspective on the goals of language description. This section focuses on the diversity of approaches characterizing missionary linguistics. The diversity is so prominent that it can be perceived quite clearly even based on a comparison of just two sample grammars (Kemp's and MacBrair's). I suggest that it is explained by an ongoing search for ways to accommodate new data into familiar forms of description, and by active interpretation of cross-linguistic differences that followed the discovery of otherness. Common practical goals define certain aspects of grammar writing, but they do not give ready solutions to the problem of cross-linguistic differences. The authors look for their own solutions, striving both to describe and to interpret the ways in which African languages are so unlike European ones.

In this section, I review different strategies the authors use to deal with situations where their data does not fit well into the familiar categories of the Graeco-Latin grammar model. I distinguish between three common types of this situation. First, the language may lack some of the familiar categories; for example, it may have no equivalent of the English past tense. Second, the language may require the speaker to use some additional categories; for example, it may use causative markers where English speakers either do not mark causativity overtly or express it by periphrastic means. Third, the languages may have roughly the same categories but express them in very different ways; for example, one language may use affixes where the other one uses an adposition (in Mande languages, aspectual meanings are commonly expressed by postpositions, cf. Nikitina 2011). From the grammarian's point of view, the three types of mismatch require different solutions. As we will see below, the situations of a category lacking in one of the languages are treated differently depending on the author, the category, and the language where it is lacking (some authors are more willing to notice and interpret gaps in an African language than analogous gaps in English). Even though this inconsistency may be perceived today as a methodological deficiency, it attests to an early stage of reflection on crosslinguistic diversity. It is this reflection that ultimately informed our modern perspectives on language description, and the different approaches used by different missionaries remind us of the alternative paths that have been tried.

Missing categories

Both Kemp's and MacBrair's grammar have a special section listing paradigms for specific aspects and moods, with accompanying remarks on difficult or peculiar aspects of the grammar of verbs. Even though these sections are rather short , they are packed with observations on the "otherness" of the ways Mande verbs function.

By far the most commonly noticed type of otherness is the absence in the local language of categories familiar from the experience with European grammars. The salience of such situations is due to the translation bias of early grammars: the absence of a direct equivalent is a prominent cause of translation difficulties. To deal with the gap, the authors often suggest a translation solution, typically in the form "the language has no x, but it uses y instead":

(18) "The Mandingo has no regular participles, or gerunds, as we find them in Western languages. In the place of a present participle, the second future is sometimes used…" (MacBrair 1842: 22)

In sections on the verb, categories commonly discussed as missing are tenses/moods, participles, and (morphological) passives. The treatment of specific gaps depends on the perceived status of the category: the absence of some categories is viewed as a reason to use periphrasis; of others, as a reflection of conceptual underdevelopment. The lack of participles, for example, is assumed to be compensated for by extensive use of periphrastic means:

(19) "The Mandingo has no past participle, which is a great defect in the language, as it causes a periphrasis to be frequently employed." (MacBrair 1842: 22) The fact that word order cannot be employed for coding information structure, on the other hand, is taken by the same author as evidence for underdevelopment of information structure categories (he does not consider, for example, the possibility that similar "niceties" of distinction could be marked in Mandinka by special markers or intonation):

(20) "Muso le dindingo kannu, properly seems to signify, "it is the woman loves the child"; but little attention is paid to such niceties of distinction by the natives." (MacBrair 1842: 36)

The contrast in the treatment of the absence of different categories suggests that the categories are perceived by the grammarians as inherently unequal. Some are taken to be universal (hence, if no direct equivalent can be found, the same meaning must be expressed periphrastically), while others are viewed as unnecessary (hence possibly undeveloped and completely ignored by speakers of the local language).

Curiously, the authors do not always agree on the status of a particular category, and hence differ in their interpretation of its absence. The lack of a morphological passive, for example, is described in very different terms by the two grammarians, even though the languages (in their present form at least) do not seem to differ in principled ways with respect to the marking of passives. MacBrair focuses on the periphrastic means that are available in Mandinka to encode the distinction between active and passive voice (21a). Kemp, on the other hand, does not discuss any strategies that could be used in Susu to draw that distinction, suggesting instead that active and passive readings are only distinguished in context (21b), cf. cf. [START_REF] Nikitina | Transitivity in Wan -An overview of constructions and verb classes. Rose-Juliet Anyanwu (ed.) Transitivity in African Languages[END_REF] on the phenomenon of passive lability.

(21a) "The passive voice is wanting in the Mandingo language. Suffering, or being acted upon by another agency, has no regular form of its own, but is expressed by a circumlocution after the manner of the French on…" (MacBrair 1842: 23) (21b) "Many Susoo verbs have both an active and neuter sense, which the construction only can determine…" (Kemp 1802: 19) The different treatment implies a different status of the distinction between active and passive voice. In MacBrair's interpretation, it is assumed to be indispensable, and presupposes some kind of formal encoding. In Kemp's treatment, it is described as context-dependent and not requiring any formal marking.

The difference in approach is consistent with difference in evaluation. MacBrair generally presents the absence of European categories as a defect, cf. his evaluation of the absence of familiar mood and tense distinctions:

(22) "The Mandingo verb is rather indefinite in its structure, resembling that of Eastern languages, by being destitute of those nice distinctions of mood and tense which are found in Western tongues." (MacBrair 1842: 14) Kemp, on the other hand, views the absence of some categories as a perfectly acceptable alternative to the Graeco-Latin standard, cf. the way the absence of morphological passive is described in (23):

(23) "It is certain, that, by the help of intransitive verbs, without ever using a passive voice, one who understands Susoo well, may communicate his thoughts with perfect clearness." (Kemp 1802: 19) The treatment of language-specific gaps as a normal aspect of cross-linguistic diversity later became a commonplace in academic grammars, and it is also taken for granted in modern descriptions.

Additional categories

Occasionally, the authors notice the presence in the local language of categories that do not exist in European languages. This happens first and foremost in cases where the local language makes additional morphological distinctions that are obligatory and must be respected in translations. Sometimes the authors resort to Greek or Latin to borrow a term they think accurately describes the additional category; for example, in (24) MacBrair explains the meaning of a tense-aspect category that he calls, after Greek, "aorist" (the description of the category's meaning suggests that a modern linguist would probably use the term "perfect" instead).

(24) [on aorist] "This mood has no equivalent of English; and therefore we borrow the name from the Greek verb. It properly describes an action with respect to both the past and the present, or the state of an object as it formerly existed and still exists." (MacBrair 1842: 14)

The asymmetry in treating the two types of mismatchsituations where the local language lacks a familiar category, and situations where it has categories in addition to those of European languagescan hardly be explained by a Eurocentric perspective. It is a rather natural consequence of the translation approach: it is simply easier to notice that something is missing in the language into which you translate than in the language of the original. On the other hand, the Eurocentric perspective clearly informs some of the interpretations of cross-linguistic differences. As we already saw, MacBrair tends to accompany mention of missing categories by evaluative judgments suggesting that the language is underdeveloped or somehow defective. He does not, however, provide any commentary on the opposite situation; for example, the mention of the aorist, which according to him has an equivalent in Ancient Greek but not in English, does not lead him to consider possible defects of English.

Similarly, MacBrair does not comment in any way on the fact that Mandinka has a causative marker for which English has no equivalent, even though that marker clearly allows its speakers to encode in a very simple way meanings that in English either receive no marking (e.g., to fell a tree) or must be expressed periphrastically (e.g., to make a tree fall).

(25) "Almost any verb may be transferred from simple agency, to the causing of an action to be performed by another agent, by the addition of ndi." (MacBrair 1842: 24) Neither does MacBrair dwell on the fact that Mandinka's TAM system is more complex in drawing additional distinctions based on negation or that Mandinka is richer than English in the inventory of existential verbs, and hence speakers of English ignore certain distinctions to which Mandinka speakers are well-attuned.

(26a) "The negative particles belonging to Mandingo verbs are differently employed, according to the particular part of the verb influenced by them." (MacBrair 1842: 22) (26b) "There may be said to be three substantive verbs" (MacBrair 1842: 25).

Hence, MacBrair's description of verbal categories is consistently Eurocentric in giving special treatment to cases where Mandinka neutralizes some of the distinctions familiar to an English speaker. Yet a Eurocentric approach does not characterize all of early missionary grammars. Kemp's description presents in this respect a striking contrast to MacBrair's. As was shown above, Kemp's interpretation of the absence of morphological passive referred to the role of context in making the distinction easy to draw in cases where it is important, allowing speakers to communicate with "perfect clearness." Consistent with this sympathetic attitude, when Kemp encounters in Susu temporal-aspectual categories that have no English equivalent, he interprets that difference as suggesting that Susu sometimes surpasses English in simplicity:

(27) "The word gei, which I have used in the Compound Future, enables the Susoos to form tenses in a very simple manner, to which the English language can scarcely be accommodated." (Kemp 1802: 31) The striking differences in the interpretation of mismatches illustrate various approaches to crosslinguistic diversity that were explored by individual authors at early stages of the development of missionary linguistics.

Same categories encoded differently

The last type of mismatch between African and European languages involves different formal encoding of roughly the same categories. It is the kind of mismatch that is hardly ever discussed explicitly, and its treatment is arguably the most problematic aspect of early grammars from the modern point of view. Especially problematic is the distinction between morphology and syntax. In contrast to inflectional Greek and Latin, Mande languages are predominantly isolating. Yet missionary grammarians often treat obviously analytic constructions as verb forms, by analogy with Greek and Latin. This results in confusion, and the authors are often forced to come up with non-standard solutions to reconcile the familiar form of presentation with empirical data to which it is alien.

One such solution was already discussed in Section 1; it involved the decision to dispense with paradigms, provided that the verb form remains unchanged (see Kemp's description in 3b). Another strategy is adopted by MacBrair to deal with the fact that Mande languages have no genitive case, but instead use a possessive marker. The possessive marker appears between the possessor and the head of the possessive construction (e.g., "John POSS father"), but unlike genitive inflection or the English possessive clitic, it does not attach to the preceding word. On the contrary, according to MacBrair's description, the possessive marker is pronounced as if attached to the following word, i.e. to the head of the possessive noun phrase. Addressing this unexpected behavior, MacBrair treats it as an irregularity that will be eliminated later (cf. earlier discussion of the language improvement project); for the time being, he suggests a temporary orthographic solution that would signal the wrong attachment of the "genitive case marker":

(28) "For this and other reasons, it has been thought proper to insert a hyphen between the two dependent nouns, which however may be dropped in the course of time, when the natives shall be habituated to grammatical construction." (MacBrair 1842: 5) Kemp seems to be in general more aware than MacBrair of the difference between morphological and syntactic means, just as he is more aware of cross-linguistic differences in the inventory of categories (see above). In the following passage he clearly draws a distinction between inflection and analytic construction; in this respect, again, Kemp's description sounds more modern than MacBrair's:

(29) "Few languages, however, are fitter for expressing every definite time than the Susoo, for while its verbs are not perplexed with endless inflections, by the help of auxiliary ones, any variety of tenses may be formed that language can require, or at least, that the most copious languages can express." (Kemp 1802: 18) Even after MacBrair, however, missionary grammarians continued to superimpose on isolating African languages a descriptive framework developed on the basis of inflectional languages. In Duport's grammar, adherence to the Graeco-Latin classification of valency-changing markers as affixal leads to inconsistent and sometimes absurd results. For example, treating a postposition as an affix, Duport arrives at a paradoxical notion of a detachable suffix, which follows the verb's object:

(30) "The suffix ra sometimes gives a transitive force to a verb; it is in this sense detached from it, and placed after the object; asfa ara = bring it, from fa = to come." (Duport 1865: 9) Similarly, adherence to the classical conjugation model leads him to mistake free-standing subject pronouns for prefixes, and to posit "prefixed pronouns" that are againparadoxicallyseparated from the verb by objects:

(31) "The conjugation of verbs is very simple, the persons being distinguished by prefixed pronouns, participles by affixes, and differences of tense by auxiliaries <…> The pronouns are always separated from the verbs, if transitive, by the object" (Duport 1865: 16)

Duport is himself not entirely comfortable with treating a Mande language as agglutinating, for he suggests that its agglutinating character is only apparent:

(32) "The agglutinating power of the language and its abundant use of prefixes and affixes give it the appearance of dealing in long words, but the roots or simple terms are either monosyllables or dissyllables" (Duport 1865: 5)

Duport lacked theoretical training that would enable him to recognize isolating structures without assuming that verbal systems must be based on inflection or affixation. The insufficient theoretical background likely has to do with a general absence of intellectual exchange between academic and missionary linguistics, cf. Irvine's description of circulation practices in late-19 th century Senegal: "Missionary texts have low circulation outside the colony, while "scientific" texts speak to a metropolitan audience (and do not seem to reach the missionaries)" (1993: 39-40). It is significant that the grammars discussed in this study do not mention any other work on the same language or on any related languages. Missionaries working on closely related languages were doing it in isolation, coming up with different solutions to the same problems.

CONCLUSION

This study revealed some ways in which early missionary grammars of Mande languages look very similar, and some ways in which they differ strikingly from each other. The similarities in presentation and interpretation of the data were argued to derive from the same practical orientation of early descriptions: not only were they intended to serve as textbooks, but they were also seen as a tool for language improvement. The diversity of approach is evident in descriptions of data that does not fit well into the Graeco-Latin framework. Individual authors put forward different solutions to the same common problemsthe mismatches between language-specific inventories of verbal categories. Some of them approach such problems in a strikingly modern way; others don't. Some adopt a radically Eurocentric approach, others highlight the advantages of the alternative type of grammar instantiated by the Mande languages. The diversity gives us a glimpse of the early stages of the development of a typology-sensitive descriptive tradition that informed the study of African languages as we presently know it. This interplay between uniformity in some aspects and diversity in others sheds light on the complex nature of the phenomenon referred to as missionary linguistics.

Many of the methodological challenges confronted by missionary grammarians were the same as the ones encountered by modern fieldworkers, yet the task of early grammarians was considerably more difficult. Not only did they not receive formal training comparable to that required of a modern descriptive linguistthey also could not rely on audio-recording equipment or on analytical tools that are nowadays taken for granted (such as the IPA transcription system). The grammars they were describing were strikingly different from those of European languages, and in the case of Mande languages, there was no local tradition of language description the missionaries could rely on. In addition, descriptions of related languages were not widely available, and did not seem to reach the missionaries. Given all that, early missionary grammars show remarkable flexibility in treating data so unlike the familiar European languages. This flexibility goes against the common assumption of the missionaries' blind reliance on the Graeco-Latin grammar tradition.

This study only addressed a very limited set of questions, and it would be incomplete if no mention was made of the theoretical aspects of the early grammars. Missionary grammars contain a wealth of ideas that go beyond language description proper. Some of the issues they raise predate modern debates in different subfields of linguistics. Observation of lexical and grammatical differences led to reflection on what is now known as linguistic relativity, and on the relationship between language and culture:

(33) "The natives of Western Africa have also little idea of the value and divisions of time; hence some ambiguity occasionally arises in relating the circumstances of an event. The author has endeavoured to fix the value of the tenses of the verb with as much precision as usage will admit of." (MacBrair 1842: vi) Differences in derivation patterns led to observations that anticipate studies in lexical typology:

(34) "Abstract terms are always formed from the concrete, never the reverse <…> So absolutely does this principle hold, that the language has no adjectives formed from nouns (as heavenly from heaven, humanus from homo)…" (Duport 1865: 6) Syntactic differences led to reflection on language evolution, in connection with contemporary philosophical thinking on the origin of parts of speech:

(35) "In examining the phrase, Charles ye dingo kannu, or, Charles a dingo le kannu, viz., Charles loves or loved the child; it would be difficult to state on what word these inserted

At its last stages, this project has received funding from the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation programme (grant agreement No 758232).

particles depend. They seem, however, to be mere disjunctive particles, to separate between the nominative and the object, which might otherwise be confused; and they are probably contracted forms of the personal pronouns. The phrase might, therefore, be rendered thus, "As for Charles, he loves the child" This accords with the forms of Eastern languages; but it gives a peculiar prominence to the pronouns, which is contrary to the imaginative schemes of some European philosophers relative to the ordinal origin of the various parts of speech." (MacBrair 1842: 31) Observations of real language use led to appreciation of usage tendencies that reflect statistical preferences rather than strict rules. Such tendenciesa still understudied subject of the modern fields of stylistics and rhetoric-are often described in terms of aesthetic preferences: (36a) "They are very fond of what I have called the Infinitive, by which they express themselves with great ease and readiness" (Kemp 1802: 32) (36b) "This particle is movable at pleasure; but its use is more elegant." (MacBrair 1842: 18)

A closer look at early missionary grammars not only helps us see the origins of some of the assumptions that are taken for granted by modern descriptive linguists; it can also remind us of the importance of pondering the otherness of different languages for developing new approaches to crosslinguistic diversity as well as for fostering new perspectives on the workings of our common human language.