Introduction

A large series of Leptotes (Cyclyrius) webbianus (Brullé, 1839) was collected in La Palma and Tenerife by Luc Manil in the early 1980s (December 1981 and July 1983) along with a lower number of male and female specimens from Gomera and Gran Canaria. Luc Manil suspected at that time that the La Palma L. webbianus [collected at the same place where Hipparchia tilosi Manil, 1984 was described] might represent a different subspecies. More recently, in July/August 2014, Xavier Mérit visited the islands of Tenerife, La Palma, La Gomera and El Hierro to collect fresh specimens of L. webbianus for DNA analysis.

Abbreviations

The following abbreviations are used in the text:

DNA Desoxyribonucleic acid, a molecule that carries the genetic information for all organisms

Ma million years

NHMW Naturhistorisches Museum, Wien

MNCN Museo Nacional de Ciencias Naturales, Madrid

Material & methods

The following material was used for phenotypic analyses: Tenerife (57 individuals: 21 ♂♂, 36 ♀♀), La Gomera (3 individuals: 2 ♂♂, 1 ♀), Gran Canaria (13 individuals: 10 ♂♂, 3 ♀♀), La Palma (44 individuals: 25 ♂♂, 19 ♀♀), and El Hierro (5 individuals: 5 ♂♂).

The width of the black margin was measured against a paper ruled in millimetre squares at space nº 2 of the males’ hindwings and at space nº 4 of the forewings.

DNA barcodes (a 658 bp fragment of the mitochondrial gene cytochrome . oxidase I) were obtained from specimens of all inhabited islands in the Canaries (Tab. 1) using standard laboratory methods (see WIEMERS & FIEDLER, 2007; DINCǍ et al., 2011; RITTER et al., 2013) using the primer pairs LepF (5’- ATT CAA CCA ATC ATA AAG ATA TTG GAA C-3’) and LepR (5’- TAA ACT TCT GGA TGT CCA AAA AAT C A-3’), or UniLepF1/UniLepR1. The latter primers are identical to LepF/LepR but have a universal T7 tail (5’ - TAA TAC GAC TCA CTA TAG GG – 3’) or T3 tail (5’ - ATT AAC CCT CAC TAA AG – 3’), respectively, attached to their 5’ end. In addition, the single available sequence from GenBank was included in the analysis.

Results

PHENOTYPIC ANALYSIS

The extension of the black margin on the dorsal side of forewings and hindwings varies between the islands. In the male specimens from Tenerife, La Gomera and El Hierro (butterflies collected by X. Mérit in 2014), the black margin of both wings typically measures less than 1 mm (0.7-1.1 mm) in width, whereas it measures approximately 1.5 mm in width in specimens from Gran Canaria and approximately 2 mm (1.8-2.3 mm) in width in the specimens from La Palma.

The postdiscal white band between veins 1 and 5 on the hindwing underside measures less than 1 mm (min: 0.8 mm - max: 0.9 mm) in width in specimens from Tenerife, La Gomera, Gran Canaria and El Hierro, compared to a width of about 1.5 mm (min: 1.3 mm – max 1.6 mm) in specimens from La Palma.

MOLECULAR ANALYSIS

The DNA analysis of the mitochondrial gene cytochrome . oxidase I (COI) confirms the differentiation of the population from La Palma with respect to the Tenerife/La Gomera lineage (minimum genetic p-distance = 1.2%, which corresponds to an age of ca. 0.5-0.8 Ma ago assuming typical substitution rates of 1.5–2.3% per Ma), as well as from the Gran Canaria population (minimum genetic p-distance = 1.5%, which corresponds to ca. 0.7-1.0 Ma ago) (Fig. 1). All analysed specimens from the other islands (Tenerife, La Gomera, and El Hierro), however, have identical COI haplotypes.

The phylogenetic relationships were inferred by using the Maximum Likelihood method based on the General Time Reversible model. The tree with the highest log likelihood (-1688.5823) is shown. The percentage of trees in which the associated taxa clustered together in a 500 replicates-bootstrap analysis is shown next to the branches. Initial tree(s) for the heuristic search were obtained automatically by applying Neighbour-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the Maximum Composite Likelihood (MCL) approach, and then selecting the topology with superior log likelihood value. A discrete Gamma distribution was used to model evolutionary rate differences among sites (5 categories (+G, parameter = 0.1)). The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. The analysis involved 11 nucleotide sequences. There were a total of 1220 positions in the final dataset. Evolutionary analyses were conducted in MEGA7 (KUMAR et al., 2016).

DESCRIPTIONS OF TWO NEW SUBSPECIES

Leptotes (Cyclyrius) webbianus palmae Mérit, Manil, Vila & Wiemers, ssp. n.

Material: Holotype ♂, SPAIN, La Palma, San Andrés y Los Sauces, Los Tilos, LP105, 370 m, 1- VIII-2014, X. Mérit leg., deposited in MNCN (fig. 2). Paratypes 1 ♂, 1 ♀, La Palma, near Los Tilos, 400 m, 25-26-VII-1983, L. Manil leg., deposited in Coll. MNCN; 6 ♂♂, 5 ♀♀, La Palma, near Los Tilos, 400 m, 25-26-VII-1983, L. Manil leg., Coll. L. Manil; 2 ♂♂, 5 ♀♀, La Palma, San Andrés y Los Sauces, Los Tilos, LP105, altitude 370 m, 1-VIII-2014, X. Mérit leg., Coll. X. Mérit; 1 ♂, 1 ♀, La Palma, Llano Molino, Barranco de la Galga, N 28º 45’ 55”-W 17º 46’ 20”, 400 m, 1-VIII-2014, X. Mérit leg., Coll. X. Mérit; 8 ♂♂, La Palma, Las Nieves, Barranco de la Madera, N 28º 42’ 14”-W 17º 47’ 20”, altitude 410 m, 31-VII-2014 / 2-VIII-2014, X. Mérit leg., Coll. X. Mérit; 1 ♀, La Palma, Barranco de Agua, altitude 200-700 m, 31-VII-1988, M. Wiemers leg., Coll. M. Wiemers; 2 ♀♀, La Palma, San Isidro, altitude 1000 m, 1-VIII-1988, M. Wiemers leg., Coll. M. Wiemers; 3 ♂♂, 1 ♀, La Palma, Barrranco de la Galga, altitude 400-800 m, 3-VIII-1988, M. Wiemers leg., Coll. M. Wiemers; 1 ♂, 1 ♀, La Palma, Barrranco de la Galga, altitude 400-800 m, ex-larva, 1-IX-1988, M. Wiemers cult., Coll. M. Wiemers. 1 ♂. used for DNA analysis, La Palma, San Andrés y Los Sauces, Los Tilos, LP105, 370 m, 1-VIII-2014, X. Mérit leg., Coll. R. Vila, code 14H868.

Description and diagnosis: The specimens from La Palma (fig. 2) differ from those from Gran Canaria, Tenerife, La Gomera and El Hierro (specimens collected in 2014) by:

1. 1. the extension of the black margin of both forewings and hindwings upperside,
2. 2. the larger extension of all the white markings, particularly the larger width of the postdiscal white band of the underside of the hindwings.
3. 3. their larger size (mean wingspan approximately 1-2 mm larger than on the other occidental islands, 1-3 mm larger than in Gran Canaria). This character is not constant.

   1. (1) In the male specimens from Tenerife, La Gomera and El Hierro (specimens collected in 2014), the black margin of both wings typically measures less than 1 mm (0.7-1.1 mm) in width, whereas it measures approximately 2 mm (1.8-2.3 mm) in width in all specimens from La Palma (red arrow).

   2. (2) The white submarginal spot near the tip of the forewing underside extends along the submarginal area till vein V5 or V4 in most specimens (green ellipse on the plate). The spot in the discal area of spaces 7-8 (black arrows) is whitish rather than brown as in the other islands. The postdiscal white band between veins 1 and 5 on the hindwing underside measures less than 1 mm in width in specimens from Tenerife, La Gomera, Gran Canaria and El Hierro (specimens collected in 2014) compared to a width of about 1.5 mm in specimens from La Palma.

   3. (3) In the female, the upperside is often fulvous rather than dark brown as in the other islands, but with a well-contrasted 2 mm dark brown marginal strip in both fore- and hindwings (red arrow).

   4. (4) The reverse side of the forewing shows often more contrasted ocelli than in most specimens originating from the other islands (blue arrows).

Distribution: This new subspecies is only known from the island of La Palma. Historical L. webbianus (extinct) collected in El Hierro more than a century ago (see discussion below and the picture on the plate) were probably closely related to ssp. palmae.

Derivatio nominis: The name of the new subspecies is derived from La Palma island where the subspecies is distributed.

Leptotes (Cyclyrius) webbianus grancanariensis Mérit, Manil, Vila & Wiemers, ssp. n.

Material: Holotype ♂, SPAIN, Gran Canaria, Cruz de Tejeda, altitude 1450 m, 8-VII-1983, L. Manil leg., deposited in MNCN (fig. 3) - Paratypes 1 ♂, Gran Canaria, Cruz de Tejeda, altitude 1450 m, 8- VII-1983, L. Manil leg., deposited in Coll. MNCN; 1 ♂♂, 1 ♀, Gran Canaria, Cruz de Tejeda, altitude 1450 m, 8-VII-1983, L. Manil leg., Coll. L. Manil; 1 ♂, 1 ♀, Gran Canaria, Pozo de la Nieves, altitude 1850 m, 09-VII-1983, L. Manil leg., Coll. L. Manil; 2 ♂♂, Gran Canaria, Fontanales, altitude 1400 m, 19-VII-1983, L. Manil leg., Coll. L. Manil; 1 ♂, Gran Canaria, Arines, altitude 1300 m, 8-VIII-1990, B. Turlin leg., Coll. X. Mérit; 2 ♂♂, used for DNA analysis, Gran Canaria, Cruz de Tejeda, altitude 1560 m, 10-V-2009, B. Acosta leg., Coll. B. Acosta; codes BA09001 and BA09002.

Description and diagnosis: The specimens from Gran Canaria (fig. 3) differ slightly from those from Tenerife, La Gomera and El Hierro (specimens collected in 2014) by the extension of the black margin of both forewings and hindwings of the dorsal face and by the width of the white line of the ventral side of the hindwings.

In the male specimens from Tenerife, La Gomera and El Hierro, the black margin of both wings typically measures less than 1 mm in width, whereas it measures approximately 2 mm in width in all specimens from La Palma and 1.5 mm in width in all specimens from Gran Canaria.

The postdiscal white band between veins 1 and 5 in specimens from Gran Canaria does not exhibit significant differences from the specimens from Tenerife and La Gomera.

The mean wingspan is slightly smaller than in the other islands, but very small specimens are particularly frequent in Gran Canaria.

Distribution: This new subspecies is known only from Gran Canaria.

Derivatio nominis: The name of the new subspecies is derived from Gran Canaria island where the subspecies is distributed.

SYSTEMATIC PLACEMENT AND SYNONYMY

Original description:

Polyommatus webbianusBrullé, 1839. Hist. Nat. Can. Ent., 1839: p. 93-94, pl. 4, fig. 1, 1a. Locus typicus: Tenerife island: Las Cañadas (above 1,400 m)

Junior synonyms:

Lycaena fortunataStaudinger, 1870. Berl. Ent. Z., 14: 99-100.

Locus typicus: Tenerife island

Polyommatus webbianus f. brunneaNordman, 1935. Commentat. Biol. 6: 6-7. Female form.

Locus typicus: La Palma island: La Caldera

CyclyriusButler, 1897. Proc. zool. Soc. Lond., 1896: 830.

Type species of CyclyriusButler, 1897 is Polyommatus webbianus Brullé

FOX et al., (1965) synonymized Cyclyrius with Leptotes Scudder, 1876, but this suggestion was not followed by most subsequent authors who continued to use Cyclyrius or the misspelling Cyclirius (but see VIVES MORENO, 2014). We provisionally use Cyclyrius as subgenus of Leptotes, pending further study on the phylogeny of Leptotes, and consider the following subspecies:

Leptotes (Cyclyrius) webbianus webbianus (Brullé, 1839), from Tenerife (fig. 4), La Gomera and El Hierro (current population)

Leptotes (Cyclyrius) webbianus palmae Mérit, Manil, Vila & Wiemers, ssp. n., from La Palma

Leptotes (Cyclyrius) webbianus grancanariensis Mérit, Manil, Vila & Wiemers, ssp. n., from Gran Canaria

Discussion

INTER-ISLAND DIFFERENTIATION OF LEPTOTES (CYCLYRIUS) WEBBIANUS

According to VAN DEN BOGAARD (2013), the Canary Islands formed between 23 Ma (Fuerteventura) and 1.1 Ma (El Hierro), and lie on the oldest hotspot track in the Atlantic Ocean, which dates back to the Late Jurassic. With 15 Ma, Gran Canaria is the oldest of the western islands, followed by Tenerife and La Gomera (12 and 11 Ma, respectively). With an age of 1.7 Ma, La Palma is only slightly older than El Hierro. Our genetic analysis does not rule out any sequence of colonization events, but is consistent with the scenario that one of the older islands (such as Gran Canaria, Tenerife or La Gomera) was colonized first, and the species then spread to the other islands in a stepwise fashion. The current spread of Leptotes pirithous in the Canary Islands (WIEMERS et al., 2013) shows that such colonization can happen within a few years. The lack of differentiation between populations on Tenerife and La Gomera probably indicates persistent gene flow between these islands. This is unsurprising considering the relatively small distance between them (less than 30 km). Additionally, it seems plausible that specimens of Leptotes (Cyclyrius) webbianus can frequently be blown over by trade winds to La Gomera from the high altitude plateau of Las Cañadas del Teide, where the species can be extremely abundant (WIEMERS 1995b).

LEPTOTES (CYCLYRIUS. WEBBIANUS ON EL HIERRO - AN INTERESTING CASE OF LOCAL EXTINCTION AND RECOLONIZATION

Leptotes (Cyclyrius) webbianus was discovered on El Hierro in 1889 by Simony (REBEL & ROGENHOFER, 1894) by at least three male samples deposited in the NHMW (Fig. 5). The collected specimens show a black margin on the forewings and hindwings upperside with a very similar width as the specimens from La Palma. Moreover, the white line on the verso of the hindwings also shows a similar pattern to those from La Palma. WIEMERS (1995b) reported the existence of these specimens as the only records known from El Hierro, but was unfortunately unable to see them at that time. No further records are known from El Hierro until MÉRIT (2015) ‘rediscovered’ it in 2014. Interestingly, the newly found specimens display a black margin and white line similar to the specimens from Tenerife and La Gomera. The DNA analysis also supports the hypothesis of a recent recolonization of El Hierro by specimens from Tenerife or La Gomera. It is very likely that the former population discovered by Simony disappeared for unknown reasons and that a new population originating from Tenerife or La Gomera recolonized the island. Unfortunately, the samples collected in 1889 are probably too old for DNA analyses, hence we cannot conclude whether the former population could have been close or identical to the newly described Leptotes (Cyclyrius) webbianus palmae.

Acknowledgments

We are grateful to Mr. Gorgonio Díaz Reyes from the Consejería de Medio Ambiente of the Canary Islands and Mr. Félix Manuel Medina, for La Palma, for the permits issued to collect butterflies. Funding for sequencing the DNA was provided by the Spanish Ministerio de Economía y Competitividad (Project CGL2013- 48277-P). We thank Benedicto Acosta-Fernández and Peter Russell for providing samples for DNA analyses and Brigitte Gottsberger (University of Vienna) for assistance in the laboratory.

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Author notes