Sheng-Hai Wu

Extinction vulnerability of tropical montane endemism from warming and upslope displacement: a preliminary appraisal for the highest massif in Madagascar

One of the predicted biological responses to climate warming is the upslope displacement of species distributions. In the tropics, because montane assemblages frequently include local endemics that are distributed close to summits, these species may be especially vulnerable to experiencing complete habitat loss from warming. However, there is currently a dearth of information available for tropical regions. Here, we present a preliminary appraisal of this extinction threat using the herpetological assemblage of the Tsaratanana Massif in northern Madagascar (the islands highest massif), which is rich with montane endemism. We present meteorological evidence (individual and combined regional weather station data and reanalysis forecast data) for recent warming in Madagascar, and show that this trend is consistent with recent climate model simulations. Using standard moist adiabatic lapse rates, these observed meteorological warming trends in northern Madagascar predict upslope species displacement of 17–74 m per decade between 1993 and 2003. Over this same period, we also report preliminary data supporting a trend for upslope distribution movements, based on two surveys we completed at Tsaratanana. For 30 species, representing five families of reptiles and amphibians, we found overall mean shifts in elevational midpoint of 19–51 m upslope (mean lower elevation limit 29–114 m; mean upper elevation limit −8 to 53 m). We also found upslope trends in mean and median elevational observations in seven and six of nine species analysed. Phenological differences between these surveys do not appear to be substantial, but these upslope shifts are consistent with the predictions based on meteorological warming. An elevational range displacement analysis projects complete habitat loss for three species below the 2 °C ‘dangerous’ warming threshold. One of these species is not contracting its distribution, but the other two were not resampled in 2003. A preliminary review of the other massifs in Madagascar indicates potential similar vulnerability to habitat loss and upslope extinction. Consequently, we urgently recommend additional elevational surveys for these and other tropical montane assemblages, which should also include, when possible, the monitoring of local meteorological conditions and habitat change.

Molecular delineation of the Y-borne Sry gene in the Formosan pangolin (Manis pentadactyla pentadactyla) and its phylogenetic implications for Pholidota in extant mammals

The systematic status of Pholidota has been a matter of debate, particularly regarding the apparent inconsistency between morphological and molecular studies. The Sry gene, a master regulator of male sex determination in eutherian mammals, has not yet been used for phylogenetic analyses of extant mammals. The objective of the present study was to clone and characterize the complete gene (1300 base pairs; bp) and amino acid sequences (229 residues) of Sry from the Formosan pangolin (Manis pentadactyla pentadactyla), a member of Pholidota. The Sry amino acid identity between pangolin and other reported species ranged from 42.5% (mouse, Mus musculus) to 84.1% (European hare, Lepus europaeus). Sequence conservation was primarily in the high motility group (HMG) box (234 bp), whereas homology outside the HMG box was low. The cloned Sry was mapped to the pangolin Y chromosome by fluorescence in situ hybridization (FISH); this was confirmed to be the first Y-borne molecular marker identified in Pholidota. Based on Bayesian phylogenetic analysis for Sry HMG sequences from 36 representative taxa, including the Formosan pangolin, Pholidota was more closely related to Carnivora than to Xenarthra, consistent with the emerging molecular tree inferred from markers not located on the Y chromosome. In conclusion, this study characterized the gene structure of Sry of the Formosan pangolin and provided insights into the phylogenetic position of Pholidota.

Preimplantation and prenatal genetic diagnosis of aromatic L-amino acid decarboxylase deficiency with an amplification refractory mutation system-quantitative polymerase chain reaction

OBJECTIVES To develop a diagnostic platform for preimplantation genetic diagnosis (PGD) and prenatal genetic diagnosis (PND) to prevent births of aromatic L-amino acid decarboxylase deficiency (AADC) patients. MATERIALS AND METHODS Five Taiwanese families carrying AADC were enrolled. A novel technique, amplification refractory mutation system-quantitative polymerase chain reaction (ARMS-qPCR), was developed for both of PGD and PND. For PGD, blastomere biopsies of day-3 cleavage-stage embryos were subjected to ARMS-qPCR. Villi, cultured amniocytes, or both were used to confirm the PGD result; this approach could also be used as the sole method for PND after in vivo conception). RESULTS Unaffected live births were achieved in four of the five families, except one with ongoing PGD. The ARMS-qPCR correctly classified blastomeres (from day-3 cleavage-stage embryos) as affected (homozygous mutant), carrier (heterozygous for mutant and wild-type alleles), or normal (homozygous wild-type) within 1 working day. CONCLUSIONS To our knowledge, this is the first report of successful PGD of AADC. The molecular technique we devised (ARMS-qPCR) was applicable for PGD as well as PND of AADC. Furthermore, it has great potential for similar applications in other monogenic disorders.

Validating a rapid, real-time, PCR-based direct mutation detection assay for preimplantation genetic diagnosis.

Although co-amplification of polymorphic microsatellite markers is the current gold standard for preimplantation genetic diagnosis (PGD) of single-gene disorders (SGD), this approach can be hampered by the lack of availability of informative markers. We recently (2011) devised a novel in-house assay for PGD of aromatic L-amino acid decarboxylase deficiency, based on an amplification refractory mutation system and quantitative PCR (ARMS-qPCR). The objective of the present study was to verify ARMS-qPCR in a cohort of 20 PGD cycles with a diverse group of SGDs (15 couples at risk for 10 SGDs). Day-3 cleavage-stage embryos were subjected to biopsy and genotyping, followed by fresh embryo transfer (FET). The diagnostic rate was 82.9%; unaffected live births were achieved in 9 of 20 FET cycles (45%), with only one false negative (among 54 transferred embryos). Overall, the ARMS-qPCR had frequent allele-dropout (ADO), rendering it inappropriate as the sole diagnostic method (despite a favorable live-birth rate). Regardless, it has the potential to complement the current gold-standard methodology, especially when trophectoderm biopsy becomes a preferred option and genotyping needs to be timely enough to enable FET.

Use of a cytogenetic whole-genome comparison to resolve phylogenetic relationships among three species: implications for mammalian systematics and conservation biology.

The objective was to apply a novel modification of a genome-wide, comparative cytogenetic technique (comparative genomic hybridization, comparative genomic hybridization (CGH)), to study species belonging to the myrmecophagous (ant/termite eating) mammalian orders/superorders (Pholidota, Tubulidentata, Carnivora, and Xenarthra), as a model for other applications in mammalian systematics and conservation biology. In this study, CGH was applied to high-quality metaphase spreads of pangolin (Pholidota), using probes of sloth and canine (Xenarthra and Carnivora, respectively) genomic DNA labeled with different fluorophores, thereby facilitating analysis of the visible color spectrum on pangolin karyotypes. Our results posited that pholidotes are closer to carnivores than to xenarthrans, which confirmed the current consensus that myrmecophagy in these mammalian lineages was more likely because of homoplasy (convergent evolution) than being an ancestral character. Since the modified CGH technique used is genome-wide, has chromosome-level resolution, and does not need full genome sequencing, it has considerable potential in systematics and other fields.

Genome-Wide Gene Expression Analysis Implicates the Immune Response and Lymphangiogenesis in the Pathogenesis of Fetal Chylothorax

Fetal chylothorax (FC) is a rare condition characterized by lymphocyte-rich pleural effusion. Although its pathogenesis remains elusive, it may involve inflammation, since there are increased concentrations of proinflammatory mediators in pleural fluids. Only a few hereditary lymphedema-associated gene loci, e.g. VEGFR3, ITGA9 and PTPN11, were detected in human fetuses with this condition; these cases had a poorer prognosis, due to defective lymphangiogenesis. In the present study, genome-wide gene expression analysis was conducted, comparing pleural and ascitic fluids in three hydropic fetuses, one with and two without the ITGA9 mutation. One fetus (the index case), from a dizygotic pregnancy (the cotwin was unaffected), received antenatal OK-432 pleurodesis and survived beyond the neonatal stage, despite having the ITGA9 mutation. Genes and pathways involved in the immune response were universally up-regulated in fetal pleural fluids compared to those in ascitic fluids. Furthermore, genes involved in the lymphangiogenesis pathway were down-regulated in fetal pleural fluids (compared to ascitic fluid), but following OK-432 pleurodesis, they were up-regulated. Expression of ITGA9 was concordant with overall trends of lymphangiogenesis. In conclusion, we inferred that both the immune response and lymphangiogenesis were implicated in the pathogenesis of fetal chylothorax. Furthermore, genome-wide gene expression microarray analysis may facilitate personalized medicine by selecting the most appropriate treatment, according to the specific circumstances of the patient, for this rare, but heterogeneous disease.

Notes on Microhyla inornata Boulenger (Anura: Microhylidae) in Taiwan

Specimens of Microhyla inornata were collected at the southern tip of Taiwan. Descriptions of adults and tadpoles are presented. We show that the separation of different color morphs of the species into subspecies is probably inappropriate. Tadpoles of M. inornata can be distinguished from M. ornata in mouth morphology, tail shape, and pigmentation. Tadpoles of this species may eat macrophytic plant material in the water, regardless of their beakless mouthparts, in addition to filter feeding on microor- ganisms. Microhyla inornata breeds during and immediately after the heavy rain. Duration of development (from eggs to froglets) is less than three weeks. Thus, this species is considered an explosive breeder. Mating calls of M. inornata consist of three frequency bands at 4.4-5.7 (fundamental), 8.8-10.0, andl4.0- 15.0 (two harmonics) kHz, respectively. The occurrence of Microhyla inornata Boulen- ger in Taiwan was first documented when Bou- lenger (1909) described the new species M. stei- negeri on the basis of five specimens collected