Comparative phylogeography of mainland and insular species of Neotropical molossid bats (Molossus)

Genetic structures, historical events, habitat preferences, and geographic barriers might result in distinct patterns in insular versus mainland populations. Comparison between these two biogeographic systems provides an opportunity to investigate the relative role of isolation in phylogeographic patterns and to elucidate the importance of evolution and demographic history in population structure. Herein we use a genotype by sequencing approach (GBS) to explore population structure within three species of mastiff bats (Molossus molossus, M. coibensis, and M. milleri), which represent different ecological histories and geographical distributions in the genus to explore phylogeographic patterns and better understand the role of geographic barriers in their dispersal and gene flow. We tested the hypotheses that oceanic straits serve as barriers to dispersal in Caribbean bats and that isolated island populations are more likely to experience genetic drift and bottlenecks relative to highly connected ones, which should have different phylogeographic patterns. We show that population structures vary according to general habitat preferences, to levels of population isolation, and to historical fluctuations in climate. In our dataset, mainland geographic barriers played only a small role in isolation of lineages. However, oceanic straits posed a partial barrier to the dispersal for some populations within some species (M. milleri), but do not seem to disrupt gene flow in others (M. molossus). Lineages on distant islands undergo genetic bottlenecks more frequently than island lineages closer to the mainland, which have a greater exchange of haplotypes.

Methods
We obtained tissue samples from 54 M. molossus from South America, Middle America and the Lesser Antilles, 20 M. coibensis from South and Middle America, and 19 M. milleri from the Greater Antilles. Samples description and localities are described in Loureiro et al. (in press). Tissues samples included skeletal muscle, liver, heart, and kidney and were preserved in 95% ethanol or were frozen in liquid nitrogen upon collection of the specimen in the field. DNA extraction was conducted using Qiagen DNeasy extraction kit (Qiagen, Inc. Valencia, CA, USA) following standard protocols. Genomic DNA quality was checked by visual inspection on an agarose gel and the DNA concentration was measured using a Nanodrop spectrophotometer (Nanodrop Techinologies). We used 30 ul of DNA samples with concentrations higher than 100 ng/ul for library preparation and for the genotyping by sequencing approach following the protocol described by Elshire et al. (2011). All libraries were sequenced on an Illumina HiSeq 2000 in the Cornell Institute of Genomic Diversity (IGD). De novo genotyping was performed using the Universal Network-Enable Analysis Kit (UNEAK) pipeline, available on TASSEL 3.0 software (Bradbury et al., 2007). Quality control and filtering of the reference genotypes of each species sample were also conducted on TASSEL 3.0  (Loureiro et al., in press).

References

Bradbury, P.J., Zhang, Z., Kroon, D.E., Casstevens, T.M., Ramdoss, Y., Buckler, E.S., 2007. TASSEL: Software for association mapping of complex traits in diverse samples. Bioinformatics 23, 2633–2635. https://doi.org/10.1093/bioinformatics/btm308

Elshire, R.J., Glaubitz, J.C., Sun, Q., Poland, J.A., Kawamoto, K., Buckler, E.S., Mitchell, S.E., 2011. A robust, simple genotyping-by-sequencing (GBS) approach for high diversity species. PLoS One 6, 1–10. https://doi.org/10.1371/journal.pone.0019379

Loureiro, L. O., Engstrom, M. D., Lim, B. K. (In press) Next Comparative phylogeography of mainland and insular species of Neotropical molossid bats (Molossus). Ecology and Evolution. Submitted on July 25, 2019. 43p. ECE-2019-06-00727.

遗传结构、历史事件、生境偏好以及地理屏障，可能会导致岛屿种群与大陆种群呈现出截然不同的种群模式。对这两类生物地理系统进行比较，可为探究隔离在系统发生地理学（phylogeographic）模式中的相对作用，以及阐明演化与种群历史对种群结构的重要性提供契机。本研究采用测序分型（genotype by sequencing, GBS）技术，对犬吻蝠属（*Molossus*）内3个物种——宽耳犬吻蝠（*Molossus molossus*）、科伊巴犬吻蝠（*M. coibensis*）和米勒犬吻蝠（*M. milleri*）——的种群结构展开探究。该3个物种在该属内具有各异的生态历史与地理分布，本研究旨在揭示其系统发生地理学模式，并深入理解地理屏障在其扩散与基因流中的作用。我们验证了两项假说：其一，加勒比海地区蝙蝠的扩散会受到海洋海峡的阻隔；其二，相较于连通性较高的种群，孤立的岛屿种群更易发生遗传漂变与瓶颈效应，两类种群应呈现出不同的系统发生地理学模式。

研究结果表明，种群结构会随整体生境偏好、种群隔离程度以及历史气候波动而发生变化。在本研究的数据集中，大陆地理屏障对支系分化的隔离作用仅占较小比例。不过，海洋海峡对部分物种内的部分种群（如米勒犬吻蝠*M. milleri*）的扩散构成了部分阻隔，但对其他物种（如宽耳犬吻蝠*M. molossus*）的基因流并未产生明显阻碍。距离大陆较远的岛屿支系，其发生遗传瓶颈的频率显著高于邻近大陆的岛屿种群——后者的单倍型交流更为频繁。

## 材料与方法
我们采集了来自南美洲、中美洲以及小安的列斯群岛的54只宽耳犬吻蝠样本，来自南美与中美洲的20只科伊巴犬吻蝠样本，以及来自大安的列斯群岛的19只米勒犬吻蝠样本。样本信息与采集地点详见Loureiro等（即将刊出）的研究。组织样本涵盖骨骼肌、肝脏、心脏与肾脏，野外采集后，样本或保存于95%乙醇中，或置于液氮内冷冻保存。DNA提取采用Qiagen DNeasy提取试剂盒（美国加利福尼亚州瓦伦西亚市Qiagen公司），严格遵循标准操作流程。通过琼脂糖凝胶电泳可视化检测基因组DNA的质量，并使用Nanodrop分光光度计（Nanodrop Technologies）测定DNA浓度。我们选取浓度高于100 ng/μL的DNA样本30 μL，按照Elshire等（2011）的实验流程，完成文库构建与测序分型操作。所有文库均在康奈尔大学基因组多样性研究所（Cornell Institute of Genomic Diversity, IGD）的Illumina HiSeq 2000平台上完成测序。基于TASSEL 3.0软件（Bradbury等，2007）中的通用网络赋能分析套件（Universal Network-Enable Analysis Kit, UNEAK）流程，完成从头基因型分型。每个物种样本的参考基因型的质量控制与过滤步骤，同样在TASSEL 3.0软件中完成（Loureiro等，即将刊出）。

## 参考文献
Bradbury, P.J., Zhang, Z., Kroon, D.E., Casstevens, T.M., Ramdoss, Y., Buckler, E.S., 2007. TASSEL: 用于多样本复杂性状关联定位的软件. *Bioinformatics*, 23, 2633–2635. https://doi.org/10.1093/bioinformatics/btm308

Elshire, R.J., Glaubitz, J.C., Sun, Q., Poland, J.A., Kawamoto, K., Buckler, E.S., Mitchell, S.E., 2011. 适用于高多样性物种的高效简易测序分型（GBS）方法. *PLoS ONE*, 6, 1–10. https://doi.org/10.1371/journal.pone.0019379

Loureiro, L. O., Engstrom, M. D., Lim, B. K. (即将刊出) 新热带区犬吻蝠属（*Molossus*）大陆与岛屿物种的比较系统发生地理学研究. *Ecology and Evolution*. 2019年7月25日提交, 共43页. ECE-2019-06-00727.