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.

Variation in abundance and habitat use of the critically endangered Microcebus gerpi across its fragmented range.

A link between the abundance of species and their degree of ecological specialization has previously been suggested within the primate order. Many species of lemurs were only recently described and even basic ecological data are not yet available for them. We investigated the habitat use, abundance, and habitat characteristics of the critically endangered Microcebus gerpi and evaluated potential impacts of vegetation structure and human disturbances on variations in its abundance. We determined abundance by systematic nocturnal surveys along 13 transects that were also used for characterizing the vegetation structure in seven study sites that were widely distributed within its range. Although M. gerpi occurred in all studied lowland rainforest and littoral forest fragments in central eastern Madagascar and therefore has a higher ecological plasticity and wider distribution than previously thought, its actual Area of Occupancy is very small (339.78 km2 ) due to an extreme degree of habitat fragmentation throughout its range. M. gerpi occurred with a mean encounter rate of 3.04 individuals/km but abundance varied substantially between sites (0.75-4.5 individuals/km). Statistical modeling revealed that the cover of small- to medium-sized trees had a positive impact on the abundance of M. gerpi, whereas a composite disturbance score (CDS), formed on the basis of information on the prominence of fires, cattle, charcoal production and wood extraction inside and around the forest, had a negative impact on abundance. These results suggest that M. gerpi is slightly less threatened than expected because of its larger geographic range, but also that it responds negatively to human disturbances. These findings raise strong conservation concerns and question the long-term viability of the remaining small and isolated populations of this arboreal solitary forager.

Diversification processes in Gerp's mouse lemur demonstrate the importance of rivers and altitude as biogeographic barriers in Madagascar's humid rainforests.

Madagascar exhibits exceptionally high levels of biodiversity and endemism. Models to explain the diversification and distribution of species in Madagascar stress the importance of historical variability in climate conditions which may have led to the formation of geographic barriers by changing water and habitat availability. The relative importance of these models for the diversification of the various forest-adapted taxa of Madagascar has yet to be understood. Here, we reconstructed the phylogeographic history of Gerp's mouse lemur (Microcebus gerpi) to identify relevant mechanisms and drivers of diversification in Madagascar's humid rainforests. We used restriction site associated DNA (RAD) markers and applied population genomic and coalescent-based techniques to estimate genetic diversity, population structure, gene flow and divergence times among M. gerpi populations and its two sister species M. jollyae and M. marohita. Genomic results were complemented with ecological niche models to better understand the relative barrier function of rivers and altitude. We show that M. gerpi diversified during the late Pleistocene. The inferred ecological niche, patterns of gene flow and genetic differentiation in M. gerpi suggest that the potential for rivers to act as biogeographic barriers depended on both size and elevation of headwaters. Populations on opposite sides of the largest river in the area with headwaters that extend far into the highlands show particularly high genetic differentiation, whereas rivers with lower elevation headwaters have weaker barrier functions, indicated by higher migration rates and admixture. We conclude that M. gerpi likely diversified through repeated cycles of dispersal punctuated by isolation to refugia as a result of paleoclimatic fluctuations during the Pleistocene. We argue that this diversification scenario serves as a model of diversification for other rainforest taxa that are similarly limited by geographic factors. In addition, we highlight conservation implications for this critically endangered species, which faces extreme habitat loss and fragmentation.

Habitat and ecological niche characteristics of the elusive Hairy-eared Dwarf Lemur (Allocebus trichotis) with updated occurrence and geographic range data.

Ecological niche modeling (ENM) is particularly useful in the study of cryptic and elusive species that are only rarely seen, difficult to study in the wild, and simultaneously threatened by a multitude of anthropogenic factors. We collected presence records one such example, the understudied Hairy-eared Dwarf Lemur (Allocebus trichotis), from the literature and supplemented these with observations from surveys in previously unstudied areas to model its fundamental and realized niche throughout Madagascar. We further evaluated habitat characteristics at each sighting location to judge its conservation status and its adaptability toward habitat degradation. We found 31 presence localities in the literature and directly observed A. trichotis a further 23 times. According to the ENMs, precipitation of the driest quarter and maximum temperature of the warmest month mostly influenced its distribution, restricting it to the higher altitudes of the northeastern humid forests of Madagascar. Its fundamental niche, extent of occurrence, and area of occupancy were estimated as 65,819, 53,054, and 16,967 km² respectively. The latter, however, declined by 29.2% since 1990. On a habitat level, it was most often observed in primary forest habitats of different degradation stages (85.7%, n = 21). The fundamental niche of A. trichotis is most likely restricted by climatic factors. Although it can be found in moderately degraded forests, it is known to depend on the availability of tree holes and therefore old-growth forest structures. Ongoing deforestation and habitat fragmentation are likely to be the major threats to the survival of this species. Our ENMs provide evidence on where to intensify survey efforts on the elusive Hairy-eared Dwarf Lemurs of Madagascar.

Population genomic structure in Goodman's mouse lemur reveals long-standing separation of Madagascar's Central Highlands and eastern rainforests.

Madagascar's Central Highlands are largely composed of grasslands, interspersed with patches of forest. The historical perspective was that Madagascar's grasslands had anthropogenic origins, but emerging evidence suggests that grasslands were a component of the pre-human Central Highlands vegetation. Consequently, there is now vigorous debate regarding the extent to which these grasslands have expanded due to anthropogenic pressures. Here, we shed light on the temporal dynamics of Madagascar's vegetative composition by conducting a population genomic investigation of Goodman's mouse lemur (Microcebus lehilahytsara; Cheirogaleidae). These small-bodied primates occur both in Madagascar's eastern rainforests and in the Central Highlands, making them a valuable indicator species. Population divergences among forest-dwelling mammals will reflect changes to their habitat, including fragmentation, whereas patterns of post-divergence gene flow can reveal formerly wooded migration corridors. To explore these patterns, we used RADseq data to infer population genetic structure, demographic models of post-divergence gene flow, and population size change through time. The results offer evidence that open habitats are an ancient component of the Central Highlands, and that widespread forest fragmentation occurred naturally during a period of decreased precipitation near the last glacial maximum. Models of gene flow suggest that migration across the Central Highlands has been possible from the Pleistocene through the recent Holocene via riparian corridors. Though our findings support the hypothesis that Central Highland grasslands predate human arrival, we also find evidence for human-mediated population declines. This highlights the extent to which species imminently threatened by human-mediated deforestation may already be vulnerable from paleoclimatic conditions.

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.

Quantitative aspects of the ruminating process in giraffes (Giraffa camelopardalis) fed with different diets.

Giraffes are ruminants feeding on fresh browse and twigs in the wild, but in zoos, their diet is mainly based on alfalfa hay, grains, and pellets occasionally supplemented by twigs and foliage. These diets, which differ in composition and digestibility, affect the behavior of the animals, tooth wear patterns, and chewing efficiency. We quantified several parameters of the rumination process in ten zoo housed giraffes of different sexes and ages fed either with alfalfa hay, fresh browse, or a combination of both. Chewing during rumination was highly ritualized and specimens showed an even distribution of chewing directions during this process, which prevents uneven tooth wear and use of chewing muscles. During rumination of alfalfa hay, chewing cycles of the giraffes took on average 49 s and included 54 jaw movements compared to 37 s and 42 jaw movements during rumination of browse, respectively. Single jaw movements (measured as basic chewing rates) were on average significantly slower during rumination of alfalfa hay (alfalfa: 1.10 chews per second, browse: 1.17 chews per second) and intercycle times between two chewing cycles took significantly longer (alfalfa: 7.77 s, browse: 7.46 s). Our results clearly indicate that several rumination parameters are influenced by the type of diet.