Chromosome-length genome assemblies and cytogenomic analyses of pangolins reveal remarkable chromosome counts and plasticity.

We report the first chromosome-length genome assemblies for three species in the mammalian order Pholidota: the white-bellied, Chinese, and Sunda pangolins. Surprisingly, we observe extraordinary karyotypic plasticity within this order and, in female white-bellied pangolins, the largest number of chromosomes reported in a Laurasiatherian mammal: 2n = 114. We perform the first karyotype analysis of an African pangolin and report a Y-autosome fusion in white-bellied pangolins, resulting in 2n = 113 for males. We employ a novel strategy to confirm the fusion and identify the autosome involved by finding the pseudoautosomal region (PAR) in the female genome assembly and analyzing the 3D contact frequency between PAR sequences and the rest of the genome in male and female white-bellied pangolins. Analyses of genetic variability show that white-bellied pangolins have intermediate levels of genome-wide heterozygosity relative to Chinese and Sunda pangolins, consistent with two moderate declines of historical effective population size. Our results reveal a remarkable feature of pangolin genome biology and highlight the need for further studies of these unique and endangered mammals.

Development and characterization of 20 polymorphic microsatellite markers for the white-bellied pangolin Phataginus tricuspis (Mammalia, Pholidota).

Pangolins, or scaly anteaters, have recently been flagshiped as one of the most illegally traded mammals, and as a corollary, as potential intermediate hosts at the origin of the COVID-19 pandemic. In order to improve the traceability of their trade, we developed 20 polymorphic microsatellite loci for the white-bellied pangolin (Phataginus tricuspis), the species most frequently found on African bushmeat markets. We genotyped 24 white-bellied pangolins from the Douala market, Cameroon, originating from the Ebo forest c. 75 km north-east of Douala. The number of alleles per locus ranged from 4 to 12 (mean = 6.95), and mean observed and expected heterozygosities were 0.592 (0.208-0.875) and 0.671 (0.469-0.836), respectively. Genetic diversity was higher than that cross-estimated from microsatellite loci developed for other species of pangolins. Two loci deviated from Hardy-Weinberg equilibrium and two loci showed linkage disequilibrium. Genetic variance (PCoA) was increased with the addition of 13 pangolins of unknown origin, possibly suggesting that the Douala market is fed from differentiated source populations of white-bellied pangolins. Each of the 37 individuals had a unique multilocus genotype. The unbiased probability of identity (uPI) and the probability of identity among siblings (PIsibs) were both very low (uPI = 8.443 e-21; PIsibs = 1.011 e-07). Only five microsatellite loci were needed to reach the conservative value of PIsibs < 0.01, overall indicating a powerful discriminating power of our combined loci. These 20 newly developed microsatellite loci might prove useful in tracing the local-to-global trade of the white-bellied pangolin, and will hopefully contribute to the DNA-assisted implementation of future conservation strategies at reasonable costs.

ECHOCARDIOGRAPHIC PARAMETERS IN AFRICAN WHITE-BELLIED PANGOLINS (PHATAGINUS TRICUSPIS) WITHOUT CARDIAC DISEASE.

Relatively little is known about pangolin care and physiology, and it has become increasingly important to have a better understanding of normal health parameters for evaluation of free-ranging, rehabilitating, and managed populations. Echocardiograms were performed on six African white-bellied pangolins (Phataginus tricuspis) under professional care at a single institution. Because of the propensity of pangolins to roll up with only thick keratinized scales exposed, the pangolins were anesthetized with either isoflurane or sevoflurane. All echocardiograms were performed and evaluated by a board-certified cardiologist. None of the pangolins had any evidence of prior cardiac disease. All standard measurements were obtained. Four pangolins (66.7%) had trace to mild mitral regurgitation, six (100%) had trace to mild tricuspid regurgitation, and two (33.3%) had trace to mild pulmonic regurgitation. All examinations were determined to be normal, with no evidence of valvular structural abnormalities, cardiac dysfunction, chamber enlargement, or cardiac hypertrophy. This report broadens understanding of normal echocardiogram parameters in pangolin species and contributes techniques and baseline data for veterinarians caring for these vulnerable populations.

COMPLICATIONS ASSOCIATED WITH PREGNANCY AND PARTURITION IN AFRICAN WHITE-BELLIED PANGOLINS (PHATAGINUS TRICUSPIS).

There are no studies to date on the normal reproductive physiology of African white-bellied pangolins (Phataginus tricuspis). As a reclusive species, little is known about normal gestation, successful parturition, and potential complications during pregnancy. Ten female P. tricuspis were diagnosed as pregnant and monitored under professional care. Five developed complications during pregnancies or during parturition and are detailed in this case series. Dystocia occurred in two dams each, with malposition of a singleton fetus. Both dams were successfully treated with surgical intervention by caesarian section. Of the two individuals, one fetus was nonviable, but the other neonate survived and was reared by the dam to weaning. A third pregnant female died during pregnancy from septicemia resulting in death of the preterm fetus. The two additional dams of the five gave birth to full-term neonates. One fetus was stillborn with evidence of fetal distress, and the other died immediately after birth (perinatal death) with undetermined etiology. Based on this case series, complications associated with pregnancy occur in P. tricuspis, indicating the need for further study and close monitoring during impending parturition.