Detection of archival Phyllonorycter mines in historical herbaria
Only 1.5% (225 out of 15,009) of herbarium specimens of Tilia spp. examined from the Palearctic contained Ph. issikii leaf mines. These 225 herbarium specimens occurred in 185 geographical locations across the Palearctic, with the westernmost point in Germany (Hessen; the herbarium specimen dated by 2004) to the most eastern locations in Japan (on the island of Hokkaido; 1885–1974) (Fig. 1).
The localities where herbarium specimens of Tilia spp. carrying Phyllonorycter mines were collected in the Palearctic in the last 253 years. The dotted line divides Ph. issikii range to native (below the line) and invaded (above the line). The map was generated using ArcGIS 9.3 (Release 9.3. New York St., Redlands, CA. Environmental Systems Research Institute, http://www.esri.com/software/arcgis/eval-help/arcgis-93).
Most specimens with leaf mines (90%; 203/225) originated from Eastern Palearctic, in particular from the Russian Far East (RFE) (67.5%, 137/203) (Fig. 2a). In some cases, leaves were severely attacked, carrying up to 12 mines per leaf (as documented in the Russian Far East in 1930s–1960s). On the other hand, we found only 22 herbarium specimens with mines (10%; 22/225) from the putative invaded region in Western Palearctic, with the majority of herbarium specimens with mines (7% 15/225) from European Russia (Fig. 2b).
The presence of Phyllonorycter issikii mines in the herbarium specimens collected in the putative native (a) and invaded (b) ranges over the past 253 years (1764–2016). The number of herbarium specimens with and without mines and the percentage of the specimens with mines in each region or country from all herbarium specimens examined in a region or country (in brackets) are given next to each graph. The total number of herbarium specimens, including those with and without mines, is given for Eastern (a) and Western Palearctic (b) separately and altogether (a + b).
The average number of leaf mines per herbarium specimen found in native (5.68 ± 0.77) and invaded regions (6.09 ± 1.70) was not significantly different (Mann–Whitney U-test: U = 20,145; Z = 0.43; p = 0.43). However, the infestation rate by Ph. issikii, i.e. percentage of leaves with mines per herbarium specimen was statistically higher in the West than in the East: 35% ± 8.19 versus 23% ± 1.94 (Mann–Whitney U-test: U = 1339; Z = 2.30; p = 0.02).
Leaf mines from the East were significantly older than those from the West (Mann–Whitney U-test: U = 81; Z = − 4.4; p < 0.001). Indeed, Ph. issikii mines from the West were found on herbarium specimens collected exclusively in the last three decades (1987–2015) (Figs. 2a, S1), whereas in East Asia, they were detected in herbarium samples dating back to 1830 (Figs. 2b, S1). The oldest mines were revealed in the pressed lime leaves sampled in the RFE: in Primorsky Krai, Sikhote-Alin Nature Reserve in 1830 (one empty mine), followed by the finding in Amur Oblast (village Busse, 1.5 km from the border with the Chinese province Heilongjiang) in 1859 (the mine contained a larva that was identified as Ph. issikii by DNA barcoding). These archival mines were 191- and 162-year-old respectively, and thus they dated 133 and 104 years before Ph. issikii description from Japan (1963). The time lag between the earliest mine record on the East (1830) and that on the West (1987) was 157 years.
We found six European herbarium specimens with eleven mines of the polyphagous moth Phyllonorycter messaniella (Fig. 1; Table S1). Seven out of eleven mines were assigned to Ph. messaniella by their morphology, i.e. presence of one distinct longitude fold on the epidermis covering the mines. However, the remaining four mines were at an early stage of development and the fold was not visible therefore DNA barcoding was used to identify them (see “Results” section below).
None of the 683 North American herbarium specimens of Tilia contained Ph. issikii mines. However, we found 37 specimens (5.4%) with 88 leaf mines of Ph. lucetiella and Ph. tiliacella with the specimens’ ages ranging from 11 to 197 years old (Table S2).
Distribution of Ph. issikii in the past as inferred from herbaria data
Eastern Palearctic (putative native range)
Out of the 3549 herbarium samples examined from the Eastern Palearctic 203 (5.7%) carried characteristic leaf mines. We found Ph. issikii mines on 41 out of 940 herbarium specimens (4.4%) in China, 3 out of 68 (4.4%) in Korea, 137 out of 2202 (i.e. 6.2%) in the RFE, and 22 out of 339 (6.5%) in Japan (Fig. 2a).
In Japan, from where Ph. issikii was formally described14, the mines were found in herbaria samples collected between 1886 and 2011, from the North to the South (32°–45° N) and from the West to the East (130°–145° E) (Fig. 3a). The earliest finding of Ph. issikii mines dated back to 1886 from Hokkaido.
Past distribution of Phyllonorycter issikii in Japan (a), the Russian Far East (b), and China (d) based on findings of leaf mines in herbaria collected in 1859–2015. The village Busse, 1.5 km from the border with the Chinese province Heilongjiang, is the location of the earliest finding of Ph. issikii mines (1859) confirmed by DNA barcoding (b, c). In China (d), the provinces where typical mines were found in herbaria are shaded in gray; in the provinces marked by an asterisk, the identification of Ph. issikii archival specimens was confirmed by DNA barcoding. The dotted line shows a schematical border between the Palearctic (above the line) and the Indomalaya (below the line). The maps were generated using ArcGIS 9.3 (Release 9.3. New York St., Redlands, CA. Environmental Systems Research Institute, http://www.esri.com/software/arcgis/eval-help/arcgis-93). Photographs in Fig. (c) taken by N. Kirichenko.
In the RFE, herbarium samples with leaf mines were collected between 1830 and 2005 in 137 localities in Primorsky Krai, Khabarovsk Krai, Jewish Autonomous Oblast and Amur Oblast (Fig. 3b). The characteristic mines were also found in pressed lime specimens from the islands Russky, Popov and Askold (sampling years 1973–1998), from where Ph. issikii was not earlier documented in the literature. According to the findings of mines in herbaria, the historical range of Ph. issikii in the RFE covered a significant area reaching the latitudes of 42°–54.5° N and the longitudes of 126°–139° E. Importantly, in the RFE the mines of Ph. issikii were regularly found in the herbaria from the localities next to the border with China and North Korea (Fig. 3b,c).
In China, the typical mines on pressed lime specimens were for the first time found in 15 provinces: from Heilongjiang in the northeast to Yunnan, Guangdong and Guangxi Zhuang Autonomous Region in the south, i.e. on the territory between 47°–32° N and 105°–125° E (Fig. 3d), whereas the species was known from China only by a single record from Tianjin20. The age of the herbarium specimens on which those mines were detected in China varied from 28 to 131 years (the herbarium specimens were collected in 1890–1993) (Fig. 3d). The oldest specimens with leaf mines originated from the northeastern provinces Jilin (1896) and Heilongjiang (1902).
In Korea, only six mines were found in three lime specimens: one mine in two herbarium specimens collected in 1900–1902 during the Korean-Sakhalin expedition by the Imperial Russian Geographical Society and the other four mines on the specimen dated by 1909 without indication of the exact sampling location on the label.
Western Palearctic (putative neocolonized range)
In Western Palearctic, only 0.2% of herbarium samples examined (22/11,466) had Ph. issikii leaf mines. In Western Siberia, 1% of herbarium samples (3/314) had Ph. issikii mines, followed by European Russia and Europe (30 countries sampled), where only 0.6% (15/2696) and 0.05% (4/8450) of the specimens, respectively, had the mines (Table 1).
None of the other 11,438 herbarium specimens collected before 1987 showed any leaf mines. In European Russia leaf mines were found in herbarium specimens collected between 1987 and 2015 in nine regions: from Leningrad Oblast on the north west to Chelyabinsk Oblast on the east (on the border between Asia and Europe) (Table 1). In Central Europe, in particular Austria, Germany, Italy, and Slovakia, archival leaf mines were found in herbarium specimens sampled between 2006 and 2015 (Table 1). In Western Siberia, the mines were found in pressed limes sampled in Khanty-Mansi Autonomous Okrug and Tyumen Oblast in 2007–2015 (Table 1).
Host plant use and level of attack of Ph. issikii in archival samples
We found archival leaf mines of Ph. issikii feeding on 18 different Tilia species in the putative native region of East Asia and only on two species of Tilia (T. cordata and T. platyphyllos) in the putative invaded area in the Western Palearctics (Fig. S2, Table S3).
In the East, 33% of all herbarium specimens with mines were found on T. amurensis (74/225) followed by T. taquetii (19%, i.e. 42/225) and T. mandshurica (11%, 24/225) (Fig. S2, Table S3). Occasionally, mines were detected on other 15 different East Asian lime species (Fig. S2, Table S3). We documented Ph. issikii mines on five novel host plants in China: T. chinensis, T. intonsa, T. leptocarya, T. miqueliana, and T. paucicostata (Fig. S2). Among these five species, T. leptocarya is presently considered as a synonym of T. endochrysea. In the West, Ph. issikii mines were found in the herbarium specimens sampled on T. cordata (15/225, i.e. 7%) and T. platyphyllos. In European Russia and Western Siberia, they were revealed only on T. cordata (7/225, i.e. 3%) (Fig. S2).
DNA barcoding of archival samples
DNA barcodes were obtained for 88 out of 93 (i.e. 95%) archival larvae and pupae of Phyllonorycter species dissected from 7 up to 162-year-old lime herbarium (Table S4). The remaining five archival larvae and pupae (four 74–125-year-old specimens from the Palearctic, and one 171-year-old specimen from the Nearctic) failed to produce sequences. Among 88 sequenced specimens 73 specimens originated from the Palearctic (dated from 1859–2014) and 15 specimens were from the Nearctic (dated from 1894–2010) (Table S4).
The length of recovered COI sequences ranged from 120 base pairs to 658 bp (Fig. 4, Table S5).
The relationship between the length of sequenced COI mtDNA fragment and the age of the archival Phyllonorycter specimens dissected from the mines in herbaria sampled in the Palearctic and Nearctic between 1850 and 2016. A linear regression is shown in the figure; y = − 2.4x + 630, R2 = 0.32, N = 93, p < 0.05. The dashed frame indicates the 71 archival specimens (from 7 to 162 years old) with relatively long sequences (> 60% of the total length); eight samples of over one century old that were successfully sequenced are highlighted within the grey rectangle.
The sequence length for the archival insect specimens was negatively and significantly correlated with the specimen age (R2 = 0.32, N = 93, p < 0.05). Relatively long sequences (> 60% of the total length, i.e. the sequenced length > 400 bp) were obtained for 71 archival specimens that were between 7 and 162 years old (Fig. 4, the points in dashed frame) (Table S4). Nine of these 71 specimens were over one century old (106–162-year-old): eight originated from the Palearctic and one from the Nearctic (Fig. 4, the points in gray cloud).
In the Palearctic, the oldest successfully DNA barcoded Ph. issikii specimen (obtained sequence length 408 bp) was a 162-year-old larva dissected from the leaf mine on Tilia amurensis from the RFE (village Busse, Amur Oblast, the year 1859), sequence ID LMINH119-19 (Fig. 5, Table S5). In the Nearctic, the oldest sequenced specimen (obtained sequence length 658 bp) was 127-year-old larva of Ph. tiliacella on T. americana from USA, Pennsylvania (Fig. 5, Table S5).
A maximum likelihood tree of 81 COI sequences of Phyllonorycter spp. Overall, 71 archival sequenced specimens were dissected from herbaria collected in the Palearctic and the Nearctic in 1859–2014 and ten specimens (highlighted in blue) originated from the modern range20. The tree was generated with the K2P nucleotide substitution model and bootstrap method (2500 iterations), p < 0.05. Each specimen is identified by its unique Process ID | country | region | host plant | sampling year | GenBank accession number (for modern sequences) or an indication of the source of the material (HERBARIUM) for archival specimens. Each genetic cluster is specified by its Barcode Index Number (BIN) (given next to each cluster). Branch lengths are proportional to the number of substitutions per site.
The 71 archival sequences represented five distinct genetic clusters, each corresponding to a unique BIN (Fig. 5).
Among them, two BINs with a Palearctic distribution were identified as Ph. issikii (number of specimens, N = 50, from 1859–1981) and Ph. messaniella (N = 2, from 1927 and 1942), and two BINs from the Nearctic were determined as Ph. lucetiella (N = 13, 1941–2010) and Ph. tiliacella (N = 2, 1894 and 1960). The fifth BIN was formed by a putative new Tilia-feeding species from the RFE and Japan20 (N = 4, 1987–1997) (Fig. 5), showing a minimum pairwise K2P distance of 4.79 with P. issikii (Table 2).
The BIN of Ph. issikii contained 50 archival specimens: 42 from the East and 8 from the West of the Palearctic. In the East, the archival specimens originated from the RFE (32 specimens from 1859–1981), China (8 specimens from 1896–1924) and Japan (2 specimens from 1905–1956) (Fig. 5). On the other hand, in the West, the archival specimens of Ph. issikii originated from Europe (2011–2015) and European part of Russia (1987–2015) (Fig. 5).
Haplotype diversity and phylogeography of Ph. issikii
Haplotype diversity in Ph. issikii past populations was higher in the East (0.93) than in the West (0.65) (Mann–Whitney U test: U = 6, Z = − 0.75, P < 0.005).
Overall, 32 haplotypes were found among 50 sequenced archival specimens of Ph. issikii (Figs. 6, 7, Tables S5, S6). All of them were detected in East Asia, including six which were shared with the West (H1, H2, H8, H13, H22, and H23) and are also present in modern specimens (Fig. 6, Table S5). The two commonest haplotypes among archival sequences were H1 (red) with 30% (15/50) sequences and H23 (green) with 14% (7/50) sequences (Figs. 6, 7). Notably, both haplotypes H1 and H23 are thought to be responsible for the invasion of the Western Palearctic dominating in the modern populations of Ph. issikii and widely found both in the Eastern and Western Palearctic20. Based on the analysis of archival herbaria, the haplotypes H13 and H23 were for the first time detected in the RFE and H22 in China, and the presence of the most distributed invasive haplotype H1 was confirmed by historical material from Japan and the RFE (Table S5) suggesting the contribution of Russia Far Eastern, Chinese and Japanese populations to the invasion of the moth westwards. Three haplotypes (H26, H28, H30) recorded in the archival Ph. issikii specimens from China and the RFE have been already known from East Asia through sequencing the insect specimens sampled in nature in twenty-first century, as per our recent study20 (Table S5).
Geographical distribution of historical and modern haplotypes of Phyllonorycter issikii found across the Palearctic. The data on the haplotype diversity of Ph. issikii in the modern range is in agreement with our previous study20. Each pie chart represents a country, except Russia where nearest locations are merged into one pie chat. Each color of pie charts refers to one of the haplotypes found across the Palearctic; the haplotypes sequenced in archival insects are shown in 10 pie charts with grey callouts. Six haplotypes are present both in the East and West Palearctic and are found in both modern and archival samples (see legend); the historical haplotypes that are not present in the modern range are counted altogether into one sector of a circle diagram with line shading. The exact geographical location of historical haplotypes can be found in Tables S5, S6. The map was generated using ArcGIS 9.3 (Release 9.3. New York St., Redlands, CA. Environmental Systems Research Institute, http://www.esri.com/software/arcgis/eval-help/arcgis-93).
Median haplotype network of Phyllonorycter issikii in the Palearctic based on sequencing of 50 archival specimens dissected from historical herbarium collected between 1859 and 2014. Different colors correspond to nine haplotypes (H1, H2, H8, H13, H22, H23, H26, H28, H30) that are also known from modern Ph. isskii specimens20. Historical unique haplotypes (documented for the first time from archival specimens) are indicated by numbers U1–U23 next to the corresponding circles with shading. Empty tiny circles indicate intermediate missing haplotypes. The number of sequenced individuals is given in brackets next to each haplotype. Each line connecting the circles indicates one mutation step. Dotted rectangles indicate the two main clusters. The geographical distribution of haplotypes is given in Tables S5, S6.
In total, 23 out of 32 haplotypes were found for the first time and exclusively in archival specimens of Ph. issikii from East Asia (shown in circles with shading in Fig. 7, Table S6).
The reconstruction of the haplotype network supported the presence of two genetically differentiated clusters (A and B) of Ph. issikii in the past range (Fig. 7). Cluster A was formed by haplotypes found in both the western and eastern parts of the Palearctic, whereas Cluster B included haplotypes exclusively found in East Asia (the Russian Far East and China) (Fig. 7). A minimum of four mutations were detected between the nearest haplotypes of clusters A and B, i.e. between U6 (Cluster A) and U7 (Cluster B), and U 15 (A) and U17 (B) (Fig. 7).
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