Population genetics and evolutionary history of the endangered Eld's deer (Rucervus eldii) with implications for planning species recovery.

Eld's deer (Rucervus eldii) with three recognised subspecies (R. e. eldii, R. e. thamin, and R. e. siamensis) represents one of the most threatened cervids found in Southeast Asia. The species has experienced considerable range contractions and local extinctions owing to habitat loss and fragmentation, hunting, and illegal trade across its distribution range over the last century. Understanding the patterns of genetic variation is crucial for planning effective conservation strategies. This study investigated the phylogeography, divergence events and systematics of Eld's deer subspecies using the largest mtDNA dataset compiled to date. We also analysed the genetic structure and demographic history of R. e. eldii using 19 microsatellite markers. Our results showed that R. e. siamensis exhibits two divergent mtDNA lineages (mainland and Hainan Island), which diverged around 0.2 Mya (95% HPD 0.1-0.2), possibly driven by the fluctuating sea levels of the Early Holocene period. The divergence between R. e. eldii and R. e. siamensis occurred around 0.4 Mya (95% HPD 0.3-0.5), potentially associated with the adaptations to warm and humid climate with open grassland vegetation that predominated the region. Furthermore, R. e. eldii exhibits low levels of genetic diversity and small contemporary effective population size (median = 7, 4.7-10.8 at 95% CI) with widespread historical genetic bottlenecks which accentuates its vulnerability to inbreeding and extinction. Based on the observed significant evolutionary and systematic distance between Eld's deer and other species of the genus Rucervus, we propose to classify Eld's deer (Cervus eldii) in the genus Cervus, which is in congruent with previous phylogenetic studies. This study provides important conservation implications required to direct the ongoing population recovery programs and planning future conservation strategies.

Season dependent effects of urban environment on circadian clock of tree sparrow (Passer montanus).

Great efforts have been made recently to understand the effect(s) of urban environments on the circadian and seasonal physiology of wild animals, but the mechanisms involved remain largely unknown. Most laboratory studies and a few studies on animals in the wild suggest alterations occur in the physiological functions of organisms in urban habitats. Here, we addressed the effects of the interaction of seasons and urban environments on clock gene expression in three tissues of tree sparrows (Passer montanus). Tree sparrows (N = 30 per site per time of year) were procured from rural and urban habitats during periods corresponding to their three physiological states, i.e., June (longest photoperiod; reproductive phase), September (equinox photoperiod; refractory phase), and December (shortest photoperiod; sensitive phase). Birds (N = 5 per time per site per month) were sampled at six time points; ZT1, ZT5, ZT9, ZT13, ZT17, and ZT21 (ZT0 = sunrise time) and clock gene expression in the hypothalamus, pineal gland, and retina was studied. Our results show that there is persistence of the circadian clock in both rural and urban birds throughout the year. In urban birds Bmal1, Npas2, Per2, and Cry1 acrophases were advanced, compared to rural birds, while Clock acrophase was delayed, depending on the tissue and time of year. This difference could be because of changes in the availability, duration, and intensity of sunlight during different times of the year and/or differential photoreceptor sensitivities, differential physiological states, or a combination of all these factors. These important results reveal, for the first time in any species, season-dependent effects of an urban environment on the molecular machinery of the circadian clock.

Daily expression of clock genes in central and peripheral tissues of tree sparrow (Passer montanus).

Almost all organisms live in a fluctuating environment. To achieve synchrony with the fluctuating environment, organisms have evolved with time-tracking mechanism commonly known as biological clocks. This circadian clock machinery has been identified in almost all cells of vertebrates and categorized as central and peripheral clocks. In birds, three independent circadian clocks reside within the nervous tissues in the hypothalamus, pineal and retina, which interact with each other and produce circadian time at a functional level. There is limited knowledge available of the molecular clockwork, and of integration between central and peripheral clocks in birds. Here, we studied daily expression of canonical clock genes (Bmal1, Clock, Per2, Per3, Cry1 and Cry2) and clock-controlled gene (Npas2) in all three central tissues (hypothalamus, pineal and retina) and in peripheral tissues (liver, intestine and muscle). Wild caught adult male tree sparrows were exposed to equinox photoperiod (12L:12D) for 2 weeks and after that birds were sacrificed (N = 5 per time point) at six time points (ZT1, ZT5, ZT9, ZT13, ZT17 and ZT21; ZT0 is lights on). Daily expression of clock genes was studied using qPCR. Bmal1, Clock, Per2, Per3, Cry1, Cry2 and Npas2 showed daily oscillation in all tissues except Cry2 in hypothalamus, pineal and intestine. We observed tissue-specific expression pattern for all clock and clock-controlled genes. Bmal1 transcripts expressed during early phase of night. Clock acrophase was observed during middle or late day time in the central clock while during early-to-middle phase of night in peripheral tissues. Npas2 expression pattern was similar to Bmal1. Per genes peaked either late at night or early during day time. However, Cry genes were peaked either at late day time (Cry1in retina, liver and intestine; Cry2 in liver and intestine) or at early night phase (Cry1 in hypothalamus, pineal and muscle; Cry2 in hypothalamus, pineal, retina and muscle). Our results are consistent with the autoregulatory circadian feedback loop, and suggest a conserved tissue-level circadian time generation in tree sparrows. Change in peak expression timing of these genes in different tissues implicates tissue-specific contribution of individual clock genes in the circadian time generation.