Anthropogenic impacts drive niche and conservation metrics of a cryptic rattlesnake on the Colorado Plateau of western North America.

Ecosystems transition quickly in the Anthropocene, whereas biodiversity adapts more slowly. Here we simulated a shifting woodland ecosystem on the Colorado Plateau of western North America by using as its proxy over space and time the fundamental niche of the Arizona black rattlesnake (Crotalus cerberus). We found an expansive (= end-of-Pleistocene) range that contracted sharply (= present), but is blocked topographically by Grand Canyon/Colorado River as it shifts predictably northwestward under moderate climate change (= 2080). Vulnerability to contemporary wildfire was quantified from available records, with forested area reduced more than 27% over 13 years. Both 'ecosystem metrics' underscore how climate and wildfire are rapidly converting the Plateau ecosystem into novel habitat. To gauge potential effects on C. cerberus, we derived a series of relevant 'conservation metrics' (i.e. genetic variability, dispersal capacity, effective population size) by sequencing 118 individuals across 846 bp of mitochondrial (mt)DNA-ATPase8/6. We identified five significantly different clades (net sequence divergence = 2.2%) isolated by drainage/topography, with low dispersal (F ST = 0.82) and small sizes (2N ef = 5.2). Our compiled metrics (i.e. small-populations, topographic-isolation, low-dispersal versus conserved-niche, vulnerable-ecosystem, dispersal barriers) underscore the susceptibility of this woodland specialist to a climate and wildfire tandem. We offer adaptive management scenarios that may counterbalance these metrics and avoid the extirpation of this and other highly specialized, relictual woodland clades.

First Report of Okra enation leaf curl virus and Associated Cotton leaf curl Multan betasatellite and Cotton leaf curl Multan alphasatellite Infecting Cotton in Pakistan: A New Member of the Cotton Leaf Curl Disease Complex.

Cotton (Gossypium hirsutum L.) is an important and widely cultivated crop in Pakistan, upon which many rely for economic security. Cotton leaf curl disease (CLCuD) is caused by a complex comprising of more than eight species in the genus Begomovirus (family Geminiviridae) with associated betasatellite and alphasatellites. During 2011, characteristic symptoms of leaf curl disease were widespread (>40%), and the whitefly Bemisia tabaci (Genn.) vector of the leaf curl complex was abundant in commercial cotton fields in Burewala, Pakistan. Symptoms included vein thickening, upward or downward leaf curling, and foliar enations. To test for the presence of a begomovirus(es), total DNA was extracted from 100 mg of symptomatic leaf tissues from five different plants (isolates CLCuDBur1 to 5) using the CTAB method (1). Total DNA extracts were used for rolling circle amplification (RCA) using TempliPhi DNA Amplification Kit (GE Healthcare). Of the five field isolates, the RCA product for only one, CLCuDBur3, digested with HindIII, produced an apparently full-length ~2.7 kb fragment, suggesting that CLCuD-Bur3 represented a distinct isolate. The 2.7-kb fragment was cloned into the plasmid vector pGEM-3Zf+ (Promega, Madison, WI). To test for the presence of associated alphasatellites and betasatellites, the PCR primers, AlphaF/R and BetaF/R (2), were used to amplify the putative 1.4-kbp molecules. The resultant 1.4-kb PCR products were ligated into the pGEMT-Easy vector and cloned. Cloned inserts for each were subjected to DNA sequencing, bidirectionally. The cloned monopartite, helper begomovirus genome (HF567945), one betasatellite (HF567946), and one alphasatellite (HF567947) sequences were determined and found to be 2,742, 1,358, and 1,376 bases long, respectively. Pairwise sequence comparisons were carried out for each using the 10 most closely related species or strains (identified in GenBank using BLASTn) using MEGA5 software. The CLCuDBur3 genome sequence shared its highest identity (99.6%) with Okra enation leaf curl virus (OELCuV) (KC019308), so CLCuDBur3 is a variant of OELCuV, a begomovirus reported previously from Abelmoschus esculentus (L.) (okra) plants in India. The betasatellite and alphasatellite shared their highest nt identity at 96 and 98.7% with Cotton leaf curl Multan betasatellite (CLCuMB) (AM774311) and Cotton leaf curl Multan alphasatellite (CLCuMA), respectively (misnamed as CLCuBuA in GenBank) (FN658728). Additionally, the HindIII-digested RCA products were analyzed by Southern blot hybridization using a DIG-labeled DNA probe specific for the intergenic region of either Cotton leaf curl Burewala virus (CLCuBuV) or OELCuV. The OELCuV, but not the CLCuBuV, probe hybridized with HindIII digested RCA products (CLCuDBur3 genome), confirming the presence of OELCuV and the absence of CLCuBuV, the latter being the most prevalent begomovirus species infecting cotton in Pakistan. This is the first report of OELCuV infecting cotton plants in Pakistan, underscoring the discovery of yet another begomovirus member of the CLCuD complex. Further, the possible co-infection of cotton by OELCuV and other recognized species of the CLCuD complex could facilitate further diversification (potentially, through recombination) and lead to the emergence of new variants with the potential to cause damage to the cotton crop in Pakistan. References: (1) J. J. Doyle and J. L. Doyle. Focus. 12:13, 1990. (2) M. Zia-Ur-Rehman et al. Plant Dis. 97:1122, 2013.

First Detection of Cotton leaf curl Burewala virus and Cognate Cotton leaf curl Multan betasatellite and Gossypium darwinii symptomless alphasatellite in Symptomatic Luffa cylindrica in Pakistan.

Cotton leaf curl disease (CLCuD) is the major plant viral constraint to cotton production on the Indian subcontinent (2). CLCuD is primarily caused by begomovirus, Cotton leaf curl Burewala virus (CLCuBuV), and Cotton leaf curl Multan betasatellite (CLCuMB). During 2011 in Burewala, Pakistan, plants in a production field of Luffa cylindrica (Ghia tori) were infested with the whitefly Bemisia tabaci (Genn.), and ~60% of the plants exhibited leaf curling and stunting symptoms, reminiscent of those caused by begomoviruses (Geminiviridae). Total DNA was extracted from five different symptomatic leaf samples using the CTAB method (1), and extracts were analyzed by Southern blot hybridization. As a probe, we used a 1.1-kbp fragment of CLCuBuV and a positive signal was obtained from all five samples. Total DNA was used as template for rolling circle amplification (RCA) using the TempliPhi DNA Amplification Kit (GE Healthcare, Little Chalfont, United Kingdom). The amplified RCA products were digested with EcoRI, and the resulting ~2.7-kbp fragments from each isolate were directionally cloned into the EcoRI digested, pGEM-3Zf+ (Promega, Madison, WI) plasmid vector. PCR was used to amplify the prospective, associated betasatellite and alphasatellite molecules using the primers BetaF5'-GGTACCGCCGGAGCTTAGCWCKCC-3' and BetaR5'-GGTACCGTAGCTAAGGCTGCTGCG-3', and AlphaF5'-AAGCTTAGAGGAAACTAGGGTTTC-3' and AlphaR5'-AAGCTTTTCATACARTARTCNCRDG-3', respectively. The putative satellite amplicons, at ~1.4 kbp each were cloned in the plasmid vector pGEMT-Easy (Promega, Madison, WI) and sequenced. BLASTn comparisons of the apparently full-length begomoviral genomes, at 2,753 nt, against the NCBI database revealed that all five isolates were most closely related to CLCuBuV (FR750321). In addition, one each of beta- and alpha-satellite were amplified from all five samples at 1,393 and 1,378 bases, respectively. The beta- and alpha-satellites were most closely related to CLCuMB (HE985228) and the Gossypium darwinii symptomless alphasatellite (GDaSA) (FR877533), respectively. Pairwise sequence comparisons of the top 10 BLASTn hits using MEGA5 indicated that the helper begomovirus shared 99.9% identity with CLCuBuV (FR750321), the most prevalent helper virus currently associated with the leaf curl complex in Pakistan. Based on the ICTV demarcation for begomoviral species at <89%, it is considered a variant of CLCuBuV. The resultant beta- and alpha-satellite sequences were 98.1% and 97.8% identical to CLCuMB (HE985228) and GDaSA (FR877533), respectively, and are the most prevalent satellites associated with the CLCuD complex in Pakistan and India (2). To our knowledge, this is first report of the CLCuBuV-CLCuMB-GDaSA complex infecting a cucurbitaceous species, and the first report of L. cylindrica as a host of the CLCuD complex. This discovery of CLCuBuV and associated satellites in a cucurbitaceous host that is widely grown in Pakistan and India where this complex infects cotton indicates that the host range of CLCuBuV is broader than expected. This new information will aid in better understanding of cotton leaf curl disease epidemiology in the current epidemic areas. References: (1) J. J. Doyle and J. L. Doyle. Focus 12:13, 1990. (2) S. Mansoor et al. Trends Plant Sci. 11:209, 2006.