Leaf beetle community data for 20 Iberian localities and associated genetic barcodes (cox1)

Field sampling and taxonomic species identification: Leaf beetle
assemblages were sampled in 20 localities along a South-North transect
(820 km) in Spain in April–June 2010. Each locality was separated from the
closest locality by a minimum of 36 km [ALC-UBG] and a maximum of 106 km
[ANC-EUM]. Sampling localities were selected to maximize the climatic
variation and spanned an altitudinal range between 120 and 1270 m a.s.l.
All localities were well preserved areas (most Natural Parks or areas with
some degree of protection) combining forests dominated by different oak
species, with shrubs and meadows. Each locality was intensively sampled by
sweeping and beating all types of vegetation, including trees, shrubs and
herbs, for 20 sampling periods of 30 min (18 sampling units in UBG).
Collecting permits were issued by the corresponding regional governments:
Junta de Andalucía (ALC, UBG, SNS), Junta de Extremadura (JCB, HOR, COR,
VER, SSP, DEL), Junta de Castilla y León (FRN, ADS, ADN, SAN, OMA, TUE)
and Xunta de Galicia (LAS, LAR, ANC, MAC, EUM). All specimens were
preserved in 100% ethanol for DNA extraction. Specimens were identified to
species level using the taxonomic monographs for the European
(Warchalowski, 2003) and the Iberian (Petitpierre, 2000) leaf beetle
faunas. When necessary, male and female genitalia were dissected and
mounted together with specimens using dimethyl-hydantoin formaldehyde
resin (DMHF). Sequence data, DNA-based species delimitation and
phylogenetic analysis: Genomic DNA was extracted from muscle tissue in the
prothorax region withWizard SV 96-well plates (Promega, UK) or with a
BioSprint 96 workstation (Qiagen, Germany). A 655 base pair region from
the 5′ end of mitochondrial cox1 was amplified with primers CO1F2
(TCTACYAATCATAAAGATATTGGTAC) and CO1R2 (ACTTCTGGATGACCAAAGAATCA) in most
cases or with standard LCO/HCO primers (Folmer et al., 1994) when the
previous primers failed. Amplification was performed with Bioline BioTaq
and the following cycling: 95 °C for 2 min, 35 cycles of 95 °C for 30 s,
40 °C for 30 s and 72 °C for 45 s, and final extension of 72 °C for 5 min.
PCR products were cleaned with 96-wellMilliporemultiscreen plates and
sequenced in both directions using ABI dye terminator sequencing. Sequence
chromatograms were assembled and manually edited using Genious 5.6. DNA
sequences are available under GenBank accession numbers KF134544 –
KF134651 and KF652242 – KF656666.

野外采样与分类学物种鉴定：2010年4月至6月，沿西班牙南北样带（820公里）的20个地点采集了叶甲群落样本。各地点间的最近距离至少为36公里[ALC-UBG]，最远距离为106公里[ANC-EUM]。采样地点的选择旨在最大化气候变异，其海拔范围介于120至1270米a.s.l.。所有地点均为受良好保护的区域（多为自然公园或具有一定保护级别的区域），涵盖了以不同栎属树种为主的森林、灌木及草地生境。每个地点通过扫网和振落法对各类植被（包括树木、灌木和草本植物）进行密集采样，共进行20个30分钟的采样时段（UBG地点为18个采样单元）。采集许可由相应的地区政府颁发：安达卢西亚自治区政府（ALC、UBG、SNS）、埃斯特雷马杜拉自治区政府（JCB、HOR、COR、VER、SSP、DEL）、卡斯蒂利亚-莱昂自治区政府（FRN、ADS、ADN、SAN、OMA、TUE）及加利西亚自治区政府（LAS、LAR、ANC、MAC、EUM）。所有标本均保存在100%乙醇中以备DNA提取。利用欧洲（Warchalowski，2003）及伊比利亚半岛（Petitpierre，2000）叶甲区系的分类学专著，将标本鉴定至物种水平。必要时，解剖雌雄生殖器，并使用二甲基乙内酰脲甲醛树脂（DMHF）与标本一同装片。序列数据、基于DNA的物种界定与系统发育分析：使用Wizard SV 96孔板（Promega公司，英国）或BioSprint 96工作站（Qiagen公司，德国）从前胸背板区域的肌肉组织中提取基因组DNA。大多数情况下，使用引物CO1F2（序列：TCTACYAATCATAAAGATATTGGTAC）和CO1R2（序列：ACTTCTGGATGACCAAAGAATCA）扩增线粒体cox1基因5′端的655碱基对区域；若上述引物扩增失败，则采用标准LCO/HCO引物（Folmer等，1994）。使用Bioline BioTaq酶进行扩增，循环条件如下：95℃预变性2分钟，35个循环（95℃变性30秒、40℃退火30秒、72℃延伸45秒），最后72℃延伸5分钟。PCR产物通过96孔Millipore多筛板纯化，并采用ABI双脱氧终止法进行双向测序。使用Genious 5.6软件组装序列色谱图并进行人工编辑。DNA序列可通过GenBank登录号KF134544–KF134651及KF652242–KF656666获取。