The evolution of host plant manipulation by insects: molecular and ecological evidence from gall-forming aphids on Pistacia M. Inbar, a, * ,1 M. Wink, b and D. Wool c a Department of Biology, University of Haifa–Oranim, Tivon 36006, Israel b Institut f€ ur Pharmazie und Molekulare Biotechnologie (IPMB), Universit€ at, Heidelberg, Abt. Biologie, Im Neuenheimer Feld 364, D-69120, Heidelberg, Germany c Department of Zoology, Tel Aviv University, Tel-Aviv 69978, Israel Received 13 July 2003; revised 7 January 2004 Available online 1 March 2004 Abstract One of the most striking characteristics of gall-forming insects is the variability in gall position, morphology, and complexity. Our knowledge of the driving forces behind the evolutionary divergence of gall types is limited. Natural enemies, competition, and behavioral constraints might be involved. We present a cladogram, based on sequences of COI and COII (1952 bp), of mito- chondrial DNA for the evolution of 14 species of gall-forming aphids (Fordinae). These insects induce five gall types with re- markable morphological variation on Pistacia spp. hosts. The parsimony cladogram divides the Fordinae into three lineages, Fordini and Baizongiini, and a third (new) sister group including the previously Fordini member, Smynthurodes betae (West). We then use ecological data to trace and explain the evolution of gall morphology. The aphids seem to have evolved gradually towards better ability to manipulate their host plant, induce stronger sinks, and gain higher reproductive success. We suggest that the an- cestral gall type was a simple, open, ‘‘pea’’-sized gall located on the leaflet midvein. Some Fordini and S. betae evolved a two-gall life cycle, inducing a new gall type on the leaflet margin. The Baizongiini improved the manipulation of their host by inducing larger galls near the midvein, with stronger sinks supporting thousands of aphids. Similar gall types are induced at similar sites on different Pistacia hosts suggesting control of the aphids on gall morphology and frequent host shifts. Thus, even extreme specialization (specific gall and host) is flexible. Ó 2004 Elsevier Inc. All rights reserved. Keywords: Extreme specialization; Gradual adaptation; Herbivore phylogeny; Host shifts; Molecular phylogeny 1. Introduction The fascinating intimate relationships between gall- forming insects and their host plants attract the atten- tion of ecologists and evolutionary biologists. One of the most striking characteristics in many groups of gall- forming insects is the variability in gall position, mor- phology, and structural complexity. Galls can be found on many host plant organs, and a given plant organ can bear various gall types (Shorthouse and Rohfritsch, 1992). Leaf galls for example, can develop on the mar- gin, blade, vein, or petiole. The galling habit probably evolved from related free- feeding insects. In thrips (Crespi and Worobey, 1998) and willow sawflies (Price, 1992; Price and Roininen, 1993), galling was probably preceded by leaf folding. In psyllids, true galls presumably developed from simple pseudogalls (Yang and Mitter, 1994). In oak galls (Stone and Cook, 1998, but see Ronquist and Liljeblad, 2001) and cerataphidine aphids (Fukatsu et al., 1994), ances- tral galls are assumed to have a single chamber or cavity, and more recent species induced multi-chambered galls. The mechanism of gall formation by insects remains unknown. However, the insects seem to control gall formation, subject to suitability and reactivity of the plant tissue (Abrahamson and Weis, 1997; Weis et al., 1988). Phylogenetic studies have suggested a tight link- age between insect relatedness and gall morphology, i.e., * Corresponding author. Fax ++ 972-4-983-2167. E-mail address: minbar@research.haifa.ac.il (M. Inbar). 1 Former surname for M. Inbar was Burstein. 1055-7903/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.ympev.2004.01.006 Molecular Phylogenetics and Evolution 32 (2004) 504–511 MOLECULAR PHYLOGENETICS AND EVOLUTION www.elsevier.com/locate/ympev