Morphological variability or inter-observer bias? A methodological toolkit to improve data quality of multi-researcher datasets for the analysis of morphological variation.

OBJECTIVES: The investigation of morphological variation in animals is widely used in taxonomy, ecology, and evolution. Using large datasets for meta-analyses has dramatically increased, raising concerns about dataset compatibilities and biases introduced by contributions of multiple researchers. MATERIALS AND METHODS: We compiled morphological data on 13 variables for 3073 individual mouse lemurs (Cheirogaleidae, Microcebus spp.) from 25 taxa and 153 different sampling locations, measured by 48 different researchers. We introduced and applied a filtering pipeline and quantified improvements in data quality (Shapiro-Francia statistic, skewness, and excess kurtosis). The filtered dataset was then used to test for genus-wide sexual size dimorphism and the applicability of Rensch's, Allen's, and Bergmann's rules. RESULTS: Our pipeline reduced inter-observer bias (i.e., increased normality of data distributions). Inter-observer reliability of measurements was notably variable, highlighting the need to reduce data collection biases. Although subtle, we found a consistent pattern of sexual size dimorphism across Microcebus, with females being the larger (but not heavier) sex. Sexual size dimorphism was isometric, providing no support for Rensch's rule. Variations in tail length but not in ear size were consistent with the predictions of Allen's rule. Body mass and length followed a pattern contrary to predictions of Bergmann's rule. DISCUSSION: We highlighted the usefulness of large multi-researcher datasets for testing ecological hypotheses after correcting for inter-observer biases. Using genus-wide tests, we outlined generalizable patterns of morphological variability across all mouse lemurs. This new methodological toolkit aims to facilitate future large-scale morphological comparisons for a wide range of taxa and applications.

Cryptic Patterns of Speciation in Cryptic Primates: Microendemic Mouse Lemurs and the Multispecies Coalescent.

Mouse lemurs (Microcebus) are a radiation of morphologically cryptic primates distributed throughout Madagascar for which the number of recognized species has exploded in the past two decades. This taxonomic revision has prompted understandable concern that there has been substantial oversplitting in the mouse lemur clade. Here, we investigate mouse lemur diversity in a region in northeastern Madagascar with high levels of microendemism and predicted habitat loss. We analyzed RADseq data with multispecies coalescent (MSC) species delimitation methods for two pairs of sister lineages that include three named species and an undescribed lineage previously identified to have divergent mtDNA. Marked differences in effective population sizes, levels of gene flow, patterns of isolation-by-distance, and species delimitation results were found among the two pairs of lineages. Whereas all tests support the recognition of the presently undescribed lineage as a separate species, the species-level distinction of two previously described species, M. mittermeieri and M. lehilahytsara is not supported-a result that is particularly striking when using the genealogical discordance index (gdi). Nonsister lineages occur sympatrically in two of the localities sampled for this study, despite an estimated divergence time of less than 1 Ma. This suggests rapid evolution of reproductive isolation in the focal lineages and in the mouse lemur clade generally. The divergence time estimates reported here are based on the MSC calibrated with pedigree-based mutation rates and are considerably more recent than previously published fossil-calibrated relaxed-clock estimates. We discuss the possible explanations for this discrepancy, noting that there are theoretical justifications for preferring the MSC estimates in this case. [Cryptic species; effective population size; microendemism; multispecies coalescent; speciation; species delimitation.].

Ecology and morphology of mouse lemurs (Microcebus spp.) in a hotspot of microendemism in northeastern Madagascar, with the description of a new species.

Delimitation of cryptic species is increasingly based on genetic analyses but the integration of distributional, morphological, behavioral, and ecological data offers unique complementary insights into species diversification. We surveyed communities of nocturnal mouse lemurs (Microcebus spp.) in five different sites of northeastern Madagascar, measuring a variety of morphological parameters and assessing reproductive states for 123 individuals belonging to five different lineages. We documented two different non-sister lineages occurring in sympatry in two areas. In both cases, sympatric species pairs consisted of a locally restricted (M. macarthurii or M. sp. #3) and a more widespread lineage (M. mittermeieri or M. lehilahytsara). Estimated Extents of Occurrence (EOO) of these lineages differed remarkably with 560 and 1,500 km2 versus 9,250 and 50,700 km2 , respectively. Morphometric analyses distinguished unambiguously between sympatric species and detected more subtle but significant differences among sister lineages. Tail length and body size were most informative in this regard. Reproductive schedules were highly variable among lineages, most likely impacted by phylogenetic relatedness and environmental variables. While sympatric species pairs differed in their reproductive timing (M. sp. #3/M. lehilahytsara and M. macarthurii/M. mittermeieri), warmer lowland rainforests were associated with a less seasonal reproductive schedule for M. mittermeieri and M. lehilahytsara compared with populations occurring in montane forests. Distributional, morphological, and ecological data gathered in this study support the results of genomic species delimitation analyses conducted in a companion study, which identified one lineage, M. sp. #3, as meriting formal description as a new species. Consequently, a formal species description is included. Worryingly, our data also show that geographically restricted populations of M. sp. #3 and its sister species (M. macarthurii) are at high risk of local and perhaps permanent extinction from both deforestation and habitat fragmentation.

Large rivers do not always act as species barriers for Lepilemur sp.

Sportive lemurs constitute a highly diverse endemic lemur family (24 species) for which many biogeographic boundaries are not yet clarified. Based on recent phylogeographic models, this study aims to determine the importance of two large rivers (the Antainambalana and Rantanabe) in northeastern Madagascar as species barriers for Lepilemur seali. The Antainambalana River was previously assumed to act as the southern border of its distribution. A total of 1,038 bp of the mtDNA of four individuals stemming from two adjacent inter-river systems south of the Antainambalana River was sequenced and compared to sequences of 22 described Lepilemur species. The phylogenetic reconstruction did not find support for either of the two rivers as species barrier for Lepilemur, as all captured individuals clustered closely with and therefore belonged to L. seali. However, a previously published sequence of an individual from a site south of our study sites belongs to a separate species. The southern boundary of L. seali must therefore be one of two large rivers further south of our study sites. The results suggest that L. seali may possess a relatively large altitudinal range that enabled this species to migrate around the headwaters of the Antainambalana and Rantanabe Rivers. Previous phylogeographic models need to be refined in order to incorporate these findings, and more species-specific altitudinal range data are urgently needed in order to fully understand the biogeographic patterns of lemurs on Madagascar.

Exceptional diversity of mouse lemurs (Microcebus spp.) in the Makira region with the description of one new species.

Although the number of described lemur species has increased considerably over the last 20 years, detailed biogeographic data are still lacking from many geographic regions, in particular in the eastern part of Madagascar. This study investigated mouse lemur species diversity in a previously unstudied Inter-River-System in the eastern Makira region. Three sites were visited and 26 individuals were sampled and characterized with 13 external morphometric measurements. Standard phylogenetic analyses were performed on the basis of sequences of three mitochondrial loci by including representatives of all other published mouse lemur species for comparison. The analyses revealed the presence of three mouse lemur species in one study site, two of which were previously undescribed. The two new species are genetically distinct and belong to the larger-bodied mouse lemur species on the island, whereas the third species, Microcebus mittermeieri, belongs to the smaller-bodied mouse lemur species. The study fully describes one of the new species. This study and other lemur inventories suggest that the Makira region is particularly rich in lemur species and the lack of any protected zone in this area should now attract the urgent attention of conservation stakeholders.