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.

The befuddling nature of mouse lemur hands and feet at Bezà Mahafaly, SW Madagascar.

The reddish-gray mouse lemur (Microcebus griseorufus) possesses striking phenotypic and behavioral variation. This project investigates differences in autopod proportions in neighboring populations of M. griseorufus from the Special Reserve at Bezà Mahafaly in southwest Madagascar. One population resides in an environment generally preferred by M. griseorufus-a spiny forest with large-trunked trees, vertically-oriented supports, and more open ground, while the other resides in a gallery forest with abundant small, often horizontal peripheral branches in high canopy. We demonstrate significant interpopulation differences in autopod morphophology despite no evidence of divergence in mitochondrial cytochrome b. We test two hypotheses regarding ultimate causation. The first, based on the Fine Branch Arborealism Hypothesis (FBAH), holds that autopod differences are related to different locomotor practices in the two environments, and the second, based on the Narrow Niche Hypothesis (NNH), holds that the observed differences reflect a relaxation (from ancestral to descendant conditions) of selective pressure for terrestrial locomotion and/or use of large, vertical supports combined with positive selection for locomoting in peripheral branch settings. Our data conform well to FBAH expectations and show some support for the NNH. Individuals from the gallery forest possess disproportionally long posterior digits that facilitate locomotion on small, flexible canopy supports while individuals from the spiny forest possess shorter posterior digits and a longer pollex/hallux that increase functional grasping diameter for large vertical supports and facilitate efficient ground locomotion. Focal individual data confirm differences in how often individuals descend to the ground and use vertical supports. We further show that predispersal juveniles, like adults, possess autopod morphologies suited to their natal forest. We explore two proximate mechanisms that could generate these cheiridial differences. The first posits an in vivo plastic response to different locomotor behaviors, the second posits differences that manifest in early development.

Unpredictable environments, opportunistic responses: Reproduction and population turnover in two wild mouse lemur species (Microcebus rufus and M. griseorufus) from eastern and western Madagascar.

Small-bodied, nocturnal mouse lemurs (Microcebus) are widespread across diverse forest habitats in Madagascar. They are strict seasonal breeders and can, depending on the habitat and species, undergo daily or prolonged torpor to minimize energy expenditure during periods of food and water scarcity. Duration of reproduction, number of litters per season and timing of births vary across individuals and species. The "polyestry-seasonality" hypothesis proposes that the duration of reproduction and number of litters per year are positively correlated with rainfall but negatively correlated with longevity, whereas the "hypervariability" hypothesis suggests that the duration of reproduction is negatively correlated with the degree of predictability of food resources. We test these hypotheses in two mouse lemur species inhabiting contrasting habitats, the brown mouse lemurs, Microcebus rufus, from Ranomafana (a less seasonal and more climatically predictable habitat) and the gray-brown mouse lemurs, M. griseorufus, from Beza Mahafaly (a more seasonal and less climatically predictable environment). We use capture/mark/recapture techniques and records of female reproductive status. We found evidence of polyestry at both study sites but faster population turnover and longer duration of the reproductive season at Beza Mahafaly. The "polyestry-seasonality" hypothesis is not supported but the "hypervariability" hypothesis could not be rejected. We conclude that reproductive output cannot be tied to climatic factors in a simple manner. Paradoxically, polyestry can be expressed in contrasting habitats: less seasonal forests where females can sustain multiple reproductive events, but also highly seasonal environments where females may not fatten sufficiently to sustain prolonged torpor but instead remain active throughout the year by relying on fallback resources. Am. J. Primatol. 77:936-947, 2015. © 2015 Wiley Periodicals, Inc.

Seasonal variation in the abundance and distribution of ticks that parasitize Microcebus griseorufus at the Bezà Mahafaly Special Reserve, Madagascar.

At Bezà Mahafaly Special Reserve (BMSR), Madagascar, mouse lemurs (Microcebus griseorufus) are parasitized by multiple species of haemaphysaline ticks. At present we know little about the role ticks play in wild lemur populations and how they can alter interspecies relationships within communities or impact host fitness. In order to better understand these dynamics at BMSR, we examined parasite-host interactions as well as the ecology of mouse lemurs and their infesting ticks, Haemaphysalis lemuris and H. sp. cf. simplex. We show that season, host sex, and habitat influence the relative abundance of ticks on mouse lemurs. Specifically, infestations occur only during the dry season (May-October), are higher in males, and are higher at the study site with the most ground cover and with greater density of large-bodied hosts. Microcebus likely experience decreased susceptibility to tick infestations during the wet season because at that time they rarely if ever descend to the ground. Similarly, male mouse lemurs have higher infestation rates than females because of the greater time they spend traveling and foraging on the ground. During the dry season, Microcebus likely serve as hosts for the tenrec tick, H. sp. cf. simplex, when tenrecs hibernate. In turn, during the wet season when mouse lemurs rarely descend to the ground, other small mammals at the reserve may serve as maintenance hosts for populations of immature ticks. The synchronous development of larvae and nymphs could present high risk for vector-borne disease in Microcebus. This study also provides a preliminary description of the ecology and life cycle of the most common lemur tick, H. lemuris.

Stable isotopes complement focal individual observations and confirm dietary variability in reddish-gray mouse lemurs (Microcebus griseorufus) from southwestern Madagascar.

We examine the ecology of reddish-gray mouse lemurs from three habitats at Beza Mahafaly Special Reserve using focal follows and stable carbon and nitrogen isotope data. Focal observations indicate dietary differences among habitats as well as sexes and seasons. Both sexes consume more arthropods during the rainy season but overall, females consume more sugar-rich exudates and fruit than males, and individuals from riparian forest consume fewer arthropods and more fruit than those in xeric or dry forest. We ask whether these observations are isotopically detectable. Isotope data support differences between seasons and sexes. Nitrogen isotope values are higher during the rainy season when lemurs consume more arthropods, and higher in males than females, particularly during the dry season. However, differences among populations inferred from focal observations are not fully supported. Lemurs from riparian forest have lower isotope values than those in xeric scrub, but isotope data suggest that lemurs from the dry forest eat the least animal matter and that focal observations overestimated dry forest arthropod consumption. Overall, our results suggest that observational and isotopic data are complementary. Isotope data can be obtained from a larger number of individuals and can quantify ingestion of animal matter, but they apparently cannot quantify the relative consumption of different sugar-rich foods. Combined focal and isotope data provide valuable insight into the dietary constraints of reddish-grey mouse lemurs, with implications for their vulnerability to future habitat change.

Biological variation in a large sample of mouse lemurs from Amboasary, Madagascar: implications for interpreting variation in primate biology and paleobiology.

A thorough knowledge of biological variation in extant primates is imperative for interpreting variation, and for delineating species in primate biology and paleobiology. This is especially the case given the recent, rapid taxonomic expansion in many primate groups, notably among small-bodied nocturnal forms. Here we present data on dental, cranial, and pelage variation in a single-locality museum sample of mouse lemurs from Amboasary, Madagascar. To interpret these data, we include comparative information from other museum samples, and from a newly collected mouse lemur skeletal sample from the Beza Mahafaly Special Reserve (BMSR), Madagascar. We scored forty dental traits (n = 126) and three pelage variants (n = 19), and collected 21 cranial/dental measures. Most dental traits exhibit variable frequencies, with some only rarely present. Individual dental variants include misshapen and supernumerary teeth. All Amboasary pelage specimens display a "reversed V" on the cap, and a distinct dorsal median stripe on the back. All but two displayed the dominant gray-brown pelage coloration typical of Microcebus griseorufus. Cranial and dental metric variability are each quite low, and craniometric variation does not illustrate heteroscedasticity. To assess whether this sample represents a single species, we compared dental and pelage variation to a documented, single-species M. griseorufus sample from BMSR. As at Amboasary, BMSR mouse lemurs display limited odontometric variation and wide variation in non-metric dental traits. In contrast, BMSR mouse lemurs display diverse pelage, despite reported genetic homogeneity. Ranges of dental and pelage variation at BMSR and Amboasary overlap. Thus, we conclude that the Amboasary mouse lemurs represent a single species - most likely (in the absence of genetic data to the contrary) M. griseorufus, and we reject their previous allocation to Microcebus murinus. Patterns of variation in the Amboasary sample provide a comparative template for recognizing the degree of variation manifested in a single primate population, and by implication, they provide minimum values for this species' intraspecific variation. Finally, discordance between different biological systems in our mouse lemur samples illustrates the need to examine multiple systems when conducting taxonomic analyses among living or fossil primates.

Incongruence between genetic and morphological diversity in Microcebus griseorufus of Beza Mahafaly.

BACKGROUND: The past decade has seen a remarkable increase in the number of recognized mouse lemur species (genus Microcebus). As recently as 1994, only two species of mouse lemur were recognized according to the rules of zoological nomenclature. That number has now climbed to as many as fifteen proposed species. Indeed, increases in recognized species diversity have also characterized other nocturnal primates--galagos, sportive lemurs, and tarsiers. Presumably, the movement relates more to a previous lack of information than it does to any recent proclivity for taxonomic splitting. Due to their nocturnal habits, one can hypothesize that mouse lemurs will show only minimal variation in pelage coloration as such variation should be inconsequential for the purposes of mate and/or species recognition. Even so, current species descriptions for nocturnal strepsirrhines place a good deal of emphasis on relatively fine distinctions in pelage coloration. RESULTS: Here, we report results from a multi-year study of mouse lemur populations from Beza Mahafaly in southern Madagascar. On the basis of morphological and pelage variation, we initially hypothesized the presence of up to three species of mouse lemurs occurring sympatrically at this locality, one of which appeared to be undescribed. Genetic analysis reveals definitively, however, that all three color morphs belong to a single recognized species, Microcebus griseorufus. Indeed, in some cases, the three color morphs can be characterized by identical mitochondrial haplotypes. CONCLUSION: Given these results, we conclude that investigators should always proceed with caution when using a single data source to identify novel species. A synthetic approach that combines morphological, genetic, geographic, and ecological data is most likely to reveal the true nature of species diversity.