A new species of bromeliad-feeding Cephaloleia Chevrolat (Coleoptera, Chrysomelidae, Cassidinae) from Costa Rica: evidence from DNA barcodes, larval and adult morphology and insect diets

Abstract The Neotropical genus Cephaloleia Chevrolat (Coleoptera: Chrysomelidae: Cassidinae) includes 214 species distributed from the south of Mexico to Argentina. Cephaloleia beetles feed mostly on plants from the order Zingiberales. The interactions between Cephaloleia beetles and their Zingiberales host plants is proposed as one of the oldest and most conservative associations. Here we describe a new species of Cephaloleia (Cephaloleia kuprewiczae sp. n.) that feeds on two species of bromeliads (Pitcairnia arcuata and Pitcairnia brittoniana, Bromeliaceae: Pitcairnioideae). Cephaloleia kuprewiczae was previously described as Cephaloleia histrionica. This study includes evidence from DNA barcodes (COI), larval and adult morphology and insect diets that separates Cephaloleia kuprewiczae from Cephaloleia histrionica as a new species.


Introduction
The Neotropical genus Cephaloleia Chevrolat (Coleoptera: Chrysomelidae: Cassidinae) includes 214 species distributed from the south of Mexico to Argentina (Staines and García-Robledo 2014). Cephaloleia beetles are also known as the "rolled-leaf beetles" because larvae and adults of the majority of Cephaloleia species feed on the scroll formed by the young leaves of their hosts. Cephaloleia beetles feed mostly on plants from the order Zingiberales. The interactions between Cephaloleia beetles and their Zingiberales host plants is one of the oldest and most conservative insect-host plant associations (García-Robledo and Staines 2008).
Two species of Cephaloleia are known to complete their life cycle on plants in the families Arecaceae and Orchidaceae (Urueta-Sandino 1972, Sekerka et al. 2013). Here we describe Cephaloleia kuprewiczae sp. n., a new species of Cephaloleia from a tropical montane forests in Costa Rica that feeds on plants from the family Bromeliaceae.
Individuals of this species were previously treated as Cephaloleia histrionica Baly (García-Robledo et al. 2013a). Combining DNA barcodes, records on host use and larval and adult morphologies, the objective of this study is to describe this new species and clarify the species delimitations between C. histrionica and Cephaloleia kuprewiczae sp. n.
In addition, we collected larvae and adults of Cephaloleia histrionica at two localities in the Talamanca Cordillera in Costa Rica, near the border with Panama. We selected these localities because they are the closest forests in Costa  Individuals were collected in ET-OH 95% for further morphological descriptions and DNA analyses. For adults, measurements were taken with an ocular micrometer. Pronotal length and width were taken along the midlines. Elytral width was measured at the humerus. Elytral length was measured from the base to the apex. Total length was measured from the base of the antennae to the apex of the elytra. For larva descriptions, measurements were taken with an ocular micrometer or from scanning electron microscope images. Total larval length was measured from the anterior to the posterior margins. Total width was measured at the widest point.

DNA sequencing and differences in COI sequences between C. kuprewiczae and C. histrionica
Legs of each adult and larval tissue were placed in 96-well plates. DNA extractions were performed following the protocols described in García-Robledo et al. (2013b). Amplification of the mitochondrial gene cytochrome oxidase COI was conducted in 96-well plate formats using the COI Folmer primer (García-Robledo et al. 2013b). PCR was followed by ExoSap purification. Amplified products were subjected to standard sequencing using BigDye Di-Deoxy terminator sequencing. Sequences were aligned using multiple sequence alignment with high accuracy and high throughput.
To estimate the similarity of COI sequences among individuals of C. kuprewiczae sp. n. and C. histrionica, we generated a neighbor-joining tree, estimating bootstrap support after 100 replicates. Analyses were performed using Geneious Pro V 5.6.5 (Biomatters-development-team 2012). Differences among COI sequences were estimated as the percentage of bases/residuals that are identical (DNA sequences: GenBank, accession No. KC794541-KC794652 and Suppl. material 1).

Host plants of Cephaloleia kuprewiczae sp. n. and C. histrionica
We recorded two host plant species for Cephaloleia kuprewiczae sp. n. At 700 m.a.s.l., larvae and adults of this species feed inside the scroll formed by the young rolled leaves of Pitcairnia arcuata ( Figure 1A-C). At 1500 m.a.s.l., C. kuprewiczae sp. n. feeds on Pitcairnia brittoniana ( Figure 1D). The damage produced by this herbivore differs from the typical longitudinal strip mining damage described for other Cephaloleia beetles (García-Robledo and Staines 2008) ( Figure 1E).
Cephaloleia histrionica was recorded feeding only on plants from the family Costaceae. In the tropical rain forest at Talamanca (60 m.a.s.l.), this species was collected from Costus guanaiensis. In the tropical premontane forest (1200 m.a.s.l.) this species was recorded feeding on Costus laevis and Dimerocostus strobilaceus.  Differential diagnosis. Cephaloleia kuprewiczae sp. n. is most similar to C. histrionica and in some degree to C. semivittata Baly. It can be easily distinguished from C. semivittata by its larger size, the elytral declivity beginning at puncture row 7, by antennomere 2 being ¾ the length of 1, by the depressed vertex of the head, and by the medial longitudinal impunctate area on the pronotum. It can be distinguished from C. histrionica by its rectangular shape and black pronotum (Figure 2). The suture between abdominal sterna 1 and 2 being obsolete medially, by elytral puncture row 10 being near lateral margin, by antennomere 2 being cylindrical, by the humerus not being reddish, and by the sinuate lateral margins of the pronotum.
Etymology. Named for Erin K. Kuprewicz, who discovered this species and its interaction with Pitcairnia (Bromeliaceae) host plants. The name is feminine.

Cephaloleia kuprewiczae sp. n.
Cephaloleia kuprewiczae immature stages were previously described as C. histrionica  The attachment tissue of the egg to the substrate is pale cream colored. Eggs were found attached to the inner surfaces of rolled leaves of P. arcuata and P. brittoniana. Mean egg length ± SD = 2.55 ± 0.09 mm, mean width ± SD = 1.59 ± 0.10 mm, n = 6. Larva color when alive is creamy-white becoming translucent laterally and apically, with some yellowish areas medially ( Figures 3B-E, 4A). Color when fixed in EtOH is yellowish-brown. Dorsum without medial setose ridge. Total length: 8.6-9.3 mm; width 4.6-4.9 mm (n = 4).
Dorsum. Pronotum without raised central area; micropustulate; with pale setae along lateral and apical margins; lateral and apical margins with numerous shallow sulci ( Figure 5D). Mesonotum without raised central area or carina or sulcus; micropustulate; laterally with numerous shallow sulci on expansion. Metanotum with central portion micropustulate; without carina or sulcus. Abdominal tergites 1-6 slightly narrowed in middle; without carina laterally; spiracle near basal margin; spiracles appear as spot with darker margin, orifice surrounded by five setae as in Figure 5F. Abdominal tergites 7-10 without surface plicae or carinae.

Differential diagnosis for larval stages
Larvae of C. kuprewiczae sp. n. and C. histrionica display obvious differences in shape and color (Figures 4 and 5). Larvae of C. kuprewiczae sp. n. are elongated and white ( Figure  4A), while C. histrionica larvae are more rounded and yellow ( Figure 4B). The head of C. kuprewiczae sp. n. is rounded ( Figure 5A), the head of C. histrionica larvae are flattened ( Figure 5B). The setae along dorsal ridges are absent in C. kuprewiczae sp. n. larvae ( Figure  5C) but present in C. histrionica. Larvae of these species are also easily differentiated by a series of five setae surrounding each spiracle only present in C. histrionica ( Figure 5E-F).
These two species can be unambiguously identified as they display a DNA barcode gap between 83-90% ( Figure 6A).
Using the DNA barcode COI, we correctly identified the species of all larvae included in this study ( Figure 6B). The neighbor-joining tree assigned all Cephaloleia kuprewiczae sp. n. individuals to one group. COI sequences of C. histrionica from the population in the tropical rain forest (60 m.a.s.l.) and premontane forest (1200 m.a.s.l.) in the Talamanca Cordillera are similar and were assigned to one group ( Figure 6B).

Discussion
This study combined morphological, ecological and molecular evidence to discover a new species. Larval morphology and differences in host plant orders are strong evidence that these are two different species. Molecular analyses confirmed that this complex includes at least two different species. It is important to note that with this information, we were able to reassess adult morphologies of C. kuprewiczae sp. n. and C. histrionica adults, finding obvious morphological differences between these two species ( Figure 2).
Previous studies reported two species of Cephaloleia completing their life cycles on palms and orchids. Cephaloleia vagelineata Pic larvae and adults were recorded on Elaeis guineensis Jacq., Corozo oleifera (H.B.K.) Bailey, Cocos nucifera L. (Urueta-Sandino 1972) and Astrocaryum chonta Matrius (Couturier and Kahn 1992) (Arecaceae).  Figure 6B). B Identification of Cephaloleia kuprewiczae sp. n. and C. histrionica using cytochrome oxidase I (COI) sequences. Neighbor-joining tree includes bootstrap values (%) supporting species identifications. Filled circles represent DNA sequences obtained from adults. Empty circles represent DNA sequences obtained from larvae.  Cephaloleia kuprewiczae sp. n. is a third example of diet expansion beyond the order Zingiberales in rolled-leaf beetles. Further studies are required to determine if other Cephaloleia species are also adapted to other non-Zingiberales host plants.