**1. Introduction**

Animals experience intense pressure to find food and mates while avoiding predation [1–3]. For many species, mate finding relies on signals that allow one sex to locate the other, and can integrate a variety of sensory modalities including visual, acoustic, electrical, and olfactory channels [4]. The same signals that increase detectability by mates can increase detectability by predators as well, and predators can impose intense selection both on the individuals doing the signaling, as well as on the individuals who are searching for mates [5–8]. Consequently, there is often intense selection for strategies that facilitate mate finding while minimizing exposure to predation.

**Citation:** Palmer, C.M.; Wershoven, N.L.; Martinson, S.J.; ter Hofstede, H.M.; Kress, W.J.; Symes, L.B. Patterns of Herbivory in Neotropical Forest Katydids as Revealed by DNA Barcoding of Digestive Tract Contents. *Diversity* **2022**, *14*, 152. https://doi.org/10.3390/d14020152

Academic Editor: Luc Legal

Received: 27 January 2022 Accepted: 17 February 2022 Published: 21 February 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

Many Orthopterans, including crickets, katydids, and grasshoppers, are known for their conspicuous signals [9,10]. These species often use energetically expensive acoustic signals to attract mates [11,12], with signals repeated again and again for a large portion of the day or night. Orthopteran signals often attract predators as well [13,14]. In the Neotropics, eavesdropping gleaning bats such as *Trachops cirrhosis* and *Lophostoma silvicolum* hunt katydids and other small animals by eavesdropping on the sounds that they produce [15–17]. Likely as a result of this acoustically targeted predation, many Neotropical forest katydids produce vanishingly little sound. In a survey of 16 phaneropterine katydid species (Tettigoniidae), none produced more than five seconds of sound per night, with most species emitting infrequent calls of 20–200 ms in duration [18]. Many of these katydid species have calls with carrier frequencies that are in the high audible or ultrasonic range [19], characteristics that would also cause a call to attenuate quickly [20], particularly in dense vegetation. Beyond Phaneropterinae, other katydid subfamilies also have instances of low calling rates in Neotropical forests, with some conocephaline and pseudophylline species producing less than 30 s of sound per night [21]. While many Neotropical forest katydids produce little sound, there are examples of species that call substantially more, such as *Ischnomela pulchripennis*, a species that is associated with spiny bromeliads that provide protection from predator attack [17]. However, with key exceptions [22], relatively little is known about possible associations between particular species of katydids and their host plants and how those associations may interact with signal structure or signaling strategy.

For species that produce so few acoustic signals, one of the inescapable questions is how they are able to encounter mates in a dense and structurally complex rainforest. There are multiple mechanisms that could facilitate mate finding in species that produce only seconds of long-distance advertisement signal each night. Phaneropterine katydids engage in mating duets, where the female replies to the male signal with a short tick, providing information about her presence and receptivity (reviewed in [23]). Duetting alone may be enough to allow katydids to find each other, if the female replies incentivize short-term risk taking and elevate male signaling rate. Males will also produce ticks that resemble the female reply, likely as a competitive mechanism that confounds interception by other males [24]. If males are producing sounds to jam other males, it suggests that it is common for multiple individuals to be present and interacting during mating, again reflecting effective strategies for co-localizing with conspecifics, rather than rare chance encounters between pairs in the forest.

An additional mechanism that could further facilitate mate encounter while minimizing conspicuousness to predators is host plant specificity. In some species, animals mate on or near their food resources, streamlining the encounter process [25,26]. Animals that find mates on a food resource can reduce travel time and associated predation risk, and species that gather at food resources may also be able to use less conspicuous signals that enable them to compete for nearby mates without attracting distant predators. If katydids gather on particular host plants and search for mates where they are gathered, this food-based aggregation strategy could dramatically lower the hurdle to mate finding, reducing female travel costs and predation risk and allowing effective pairing, even with rare, short duration, rapidly attenuating signals.

Host plant specialization provides opportunities and challenges. Mature tropical forests contain a diversity of vegetation, much of which is heavily protected by secondary compounds and chemically defended against most herbivores [27–29]. Herbivores respond to plant defenses with a diversity of strategies, including extreme host generalization, where they eat small quantities of many plants to minimize the impacts of each type of toxin [30,31], or host plant specialization, where they evolve to tolerate or even repurpose a particular type of chemical defense [32,33]. In most habitats, herbivorous Orthopterans consume a wide range of plants [34]. However, there are cases where Orthopterans specialize on a particular food source, with some displaying strong associations and genetic differentiation based on diet [35,36], while others demonstrate strong preferences for specific plants but accept other plant species when preferred options are not available [37].

We test the hypothesis that many Neotropical forest phaneropterines are host plant specialists, facilitating pairing and reducing the demands associated with eating a large diversity of highly defended rainforest plants. The diet specialization hypothesis predicts that all katydids of a particular species will consistently consume the same or closely related plant species across space and time. An alternative hypothesis is that katydids are more generalized in their diets, a hypothesis that would be supported by sampling multiple individuals of the same katydid species and finding that they had been eating taxonomically diverse plant species.

Katydids are renowned for their camouflage and finding them in the forest can be very challenging, particularly because many species occur in the forest canopy [38]. Because it is so difficult to observe these animals in the wild, it is also difficult to determine if they are dietary specialists. A detailed review of the literature on these katydid species [19] did not yield any published records of diet. Fortunately, DNA sequencing approaches make it possible to identify plants that are part of the diets by extracting plant material from the stomachs of captured katydids and amplifying plant DNA barcodes [39–42]. These plant DNA barcodes have been developed specifically for the research site on Barro Colorado Island in Panama and have been previously utilized to generate a community phylogeny of trees and assign plant species to fine roots collected in the soil [43,44]. Here we employ these barcodes to test diet specialization of katydids.
